Alleles of the rel gene from coryneform bacteria

ABSTRACT

An isolated mutant of a coryneform bacterium comprising a gene coding for a polypeptide having GTP-pyrophosphate kinase activity, wherein said polypeptide comprises an amino acid sequence in which one of the proteinogenic amino acids other than L-proline is present in position 38 or a corresponding or comparable position. In addition, an isolated polynucleotide encoding a polypeptide having GTP-pyrophosphate kinase enzyme activity, a vector comprising the isolated polynucleotide, a recombinant microorganism comprising the vector, and a process for preparing the recombinant coryneform bacterium is described. A method for over-expressing a GTP-pyrophosphate kinase, a method of preparing an L-amino acid, an L-lysine comprising and L-tryptophan comprising feed is also described.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.11/873,073, filed Oct. 16, 2007, which is incorporated herein byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to mutants and alleles of the rel gene ofcoryneform bacteria, which encode variants of GTP-pryophosphate kinase,and to processes for preparing amino acids, in particular L-lysine,L-tryptophan, L-proline, L-valine, L-isoleucine and L-homoserine, byusing bacteria which harbor the alleles.

2. Discussion of the Background

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.Further, the materials, methods, and examples are illustrative only andare not intended to be limiting, unless otherwise specified.

Amino acids are applied in human medicine, in the pharmaceuticalindustry, in the food industry and in animal nutrition.

Amino acids are known to be prepared by fermentation of strains ofcoryneform bacteria, in particular Corynebacterium glutamicum. Due tothe great importance, continuous efforts are made to improve theproduction processes. The processes may be improved with respect tofermentation-related measures such as, for example, stirring and oxygensupply or the composition of the nutrient media, such as, for example,sugar concentration during the fermentation, or the working-up intoproduct form, for example by means of ion exchange chromatography, orthe intrinsic performance characteristics of the microorganism itself.

The performance characteristics of said microorganisms are improved byapplying methods of mutagenesis, selection and mutant choice. Thisenables strains to be obtained which are resistant to antimetabolites orauxotrophic for metabolites which are of regulatory importance, andproduce amino acids. A known antimetabolite is the lysine analogS-(2-aminoethyl)-L-cysteine (AEC).

For some years now, methods of recombinant DNA technology have likewisebeen employed in order to improve L-amino acid-producing Corynebacteriumstrains, by amplifying individual amino acid biosynthesis genes andstudying the effect on amino acid production.

The Corynebacterium glutamicum chromosome was sequenced completely sometime ago (Kalinowski et al., Journal of Biotechnology 104, 5-25 (2003)).The Corynebacterium efficiens chromosome has likewise been sequencedpreviously (Nishio et al., Genome Res. 13 (7), 1572-1579 (2003)).

Corresponding sequence information can be found in the public databases.Suitable databases are, for example, the database of the EuropeanMolecular Biologies Laboratories (EMBL, Heidelberg, Germany andCambridge, UK), the database of the National Center for BiotechnologyInformation (NCBI, Bethesda, Md., USA), that of the Swiss Institute ofBioinformatics (Swissprot, Geneva, Switzerland), the protein InformationResource Database (PIR, Washington, D.C., USA) and the DNA Data Bank ofJapan (DDBJ, 1111 Yata, Mishima, 411-8540, Japan).

Overviews on the genetics, the metabolism and the technical industrialimportance of Corynebacterium can be found in the papers by Ikeda, byPfefferle et al. and Mueller and Huebner in the book “MicrobialProduction of L-Amino Acids” (Advances in Biochemical Engineering 79,(2003), Springer Verlag, Berlin, Germany, Editor: T. Scheper), in thespecial edition “A New Era in Corynebacterium glutamicum Biotechnology”of the Journal of Biotechnology (Volume 104 (1-3), 2003, Editor: A.Pühler and T. Tauch), and in the “Handbook of Corynebacteriumglutamicum” (Editors: L. Eggeling and M. Bott, CRC Press, Taylor &Francis Group, Boca Raton, Fla., USA, 2005).

The nucleotide sequence of the rel gene coding for GTP-pyrophosphatekinase of Corynebacterium glutamicum is accessible, inter alia in thedatabase of the National Center for Biotechnology Information (NCBI) ofthe National Library of Medicine (Bethesda, Md., USA) under the accessnumber AF038651. It can furthermore be found as sequence no. 1824 in thepatent application EP 1 108 790.

Wehmeier et al. (Microbiology 144, 1853-1862 (1998)) report, inter alia,genetic, microbiological and biochemical studies on a mutant ofCorynebacterium glutamicum ATCC 13032 which carries a deletion in therel gene.

For reasons of better clarity, SEQ ID NO:1 depicts the nucleotidesequence of the rel gene coding for GTP-pyrophosphate kinase of the wildtype of Corynebacterium glutamicum (“wild type gene”), according to theinformation of the NCBI database, and SEQ ID NO:2 or 4 depict the aminoacid sequence derived therefrom of the encoded GTP-pyrophosphate kinase.In addition, SEQ ID NO:3 indicates nucleotide sequences located upstreamand downstream. The amino acid sequence according to SEQ ID NO: 2 and 4comprises glycine in position 262. The amino acid sequence of wildtypeGTP-pyrophosphate kinase, disclosed in EP 1 108 790 comprises L-glutamicacid in position 262. The nucleotide sequence of the wildtype rel geneaccording to EP 1 108 790 is depicted in sequence SEQ ID NO:21. SEQ IDNO:22 represents the encoded amino acid sequence.

SUMMARY OF THE INVENTION

It was an object of the present invention to provide novel measures forimproving the production of amino acids, in particular L-lysine,L-tryptophan, L-proline, L-valine, L-isoleucine and L-homoserine.

This and other objects have been achieved by the present invention thefirst embodiment of which includes an isolated mutant of a coryneformbacterium comprising a gene coding for a polypeptide havingGTP-pyrophosphate kinase activity, wherein said polypeptide comprises anamino acid sequence in which one of the proteinogenic amino acids otherthan L-proline is present in position 38 or a corresponding orcomparable position.

The invention further provides an isolated polynucleotide encoding apolypeptide having GTP-pyrophosphate kinase enzyme activity, a vectorcomprising the isolated polynucleotide, a recombinant microorganismcomprising the vector, and a process for preparing the recombinantcoryneform bacterium is described.

The invention also provides a method for over-expressing aGTP-pyrophosphate kinase, a method of preparing an L-amino acid, anL-lysine-comprising and L-tryptophan-comprising feed is also described.

BRIEF DESCRIPTION OF THE DRAWINGS

The FIGURE represents a map of the plasmid pK18moBsacB_rel_P38L.

DESCRIPTION OF THE INVENTION

The invention relates to generated or isolated mutants of coryneformbacteria which preferably secrete amino acids, and which comprise a geneor allele encoding a polypeptide having GTP-pyrophosphate kinaseactivity, wherein said polypeptide comprises an amino acid sequence inwhich one of the proteinogenic amino acids other than L-proline ispresent in position 38 or a corresponding or comparable position of theamino acid sequence, preferably, L-proline is substituted withL-leucine.

Among the coryneform bacteria, preference is given to the genusCorynebacterium. Among the genus Corynebacterium, preference is given tothe following species:

-   -   Corynebacterium efficiens (strain type DSM44549),    -   Corynebacterium glutamicum (strain type ATCC13032),    -   Corynebacterium thermoaminogenes (for example the strain FERM        BP-1539), and    -   Corynebacterium ammoniagenes (strain type ATCC6871),

more preferably, to the species Corynebacterium glutamicum.

Some representatives of the species Corynebacterium glutamicum are alsoknown under different species names in the prior art. These include, forexample:

-   -   Corynebacterium acetoacidophilum ATCC13870,    -   Corynebacterium lilium DSM20137,    -   Corynebacterium melassecola ATCC17965,    -   Brevibacterium flavum ATCC14067,    -   Brevibacterium lactofermentum ATCC13869,    -   Brevibacterium divaricatum ATCC14020, and    -   Microbacterium ammoniaphilum ATCC15354.

The term “Micrococcus glutamicus” has also been used for Corynebacteriumglutamicum.

The strains of coryneform bacteria employed for the purposes of theinvention preferably already have the ability to concentrate the desiredamino acid in the cell or to secrete and accumulate it in thesurrounding nutrient medium. This is also referred to by the term “toproduce” hereinbelow. Specifically, the strains of coryneform bacteriaemployed have the ability to concentrate or accumulate=(at least) 0.25g/l, =0.5 g/l, =1.0 g/l, =1.5 g/l, =2.0 g/l, =4 g/l or =10 g/1 of thedesired amino acid in the cell or in the nutrient medium within=(no morethan) 120 hours, =96 hours, =48 hours, =36 hours, =24 hours or =12hours. The strains may be those which have been prepared by mutagenesisand selection, by recombinant DNA techniques or by a combination of bothmethods.

Examples of known representatives of L-lysine-producing or -secretingstrains of coryneform bacteria are:

-   -   Corynebacterium glutamicum DM58-1/pDM6 (=DSM4697) described in        EP 0 358 940,    -   Corynebacterium glutamicum MH20-22B (=DSM16835) described in        Menkel et al. (Applied and Environmental Microbiology 55(3),        684-688 (1989)),    -   Corynebacterium glutamicum AHP-3 (=Ferm BP-7382) described in EP        1 108 790,    -   Corynebacterium glutamicum NRRL B-11474 described in U.S. Pat.        No. 4,275,157, and    -   Corynebacterium thermoaminogenes AJ12521 (=FERM BP-3304)        described in U.S. Pat. No. 5,250,423.

Examples of known representatives of L-tryptophan-producing or-secreting strains of coryneform bacteria are:

-   -   Corynebacterium glutamicum K76 (=Ferm BP-1847) described in U.S.        Pat. No. 5,563,052,    -   Corynebacterium glutamicum BPS13 (=Ferm BP-1777) described in        U.S. Pat. No. 5,605,818, and    -   Corynebacterium glutamicum Ferm BP-3055 described in U.S. Pat.        No. 5,235,940.

Examples of known representatives of L-proline-producing or -secretingstrains of coryneform bacteria are:

-   -   Brevibacterium lactofermentum NRRL B-11421 described in U.S.        Pat. No. 4,224,409,    -   Brevibacterium flavum NRRL B-11422 described in U.S. Pat. No.        4,224,409,    -   Brevibacterium flavum FERM BP-2214 described in U.S. Pat. No.        5,294,547,    -   Corynebacterium glutamicum NRRL B-11423 described in U.S. Pat.        No. 4,224,409,    -   Corynebacterium glutamicum ATCC 21157 described in U.S. Pat. No.        4,444,885,    -   Corynebacterium glutamicum ATCC 21158 described in U.S. Pat. No.        4,444,885,    -   Corynebacterium glutamicum ATCC 21159 described in U.S. Pat. No.        4,444,885,    -   Corynebacterium glutamicum ATCC 21355 described in U.S. Pat. No.        4,444,885, and    -   Macrobacterium ammoniaphilum NRRL B-11424 described in U.S. Pat.        No. 4,224,409.

Examples of known representatives of L-valine-producing or -secretingstrains of coryneform bacteria are:

-   -   Brevibacterium lactofermentum FERM BP-1763 described in U.S.        Pat. No. 5,188,948,    -   Brevibacterium lactofermentum FERM BP-3007 described in U.S.        Pat. No. 5,521,074,    -   Corynebacterium glutamicum FERM BP-3006 described in U.S. Pat.        No. 5,521,074, and    -   Corynebacterium glutamicum FERM BP-1764 described in U.S. Pat.        No. 5,188,948.

Examples of known representatives of L-isoleucine-producing or-secreting strains of coryneform bacteria are:

-   -   Brevibacterium flavum FERM BP-760 described in U.S. Pat. No.        4,656,135,    -   Brevibacterium flavum FERM BP-2215 described in U.S. Pat. No.        5,294,547, and    -   Corynebacterium glutamicum FERM BP-758 described in U.S. Pat.        No. 4,656,135.

Examples of known representatives of L-homoserine-producing or-secreting strains of coryneform bacteria are:

-   -   Micrococcus glutamicus ATCC 14296 described in U.S. Pat. No.        3,189,526 and    -   Micrococcus glutamicus ATCC 14297 described in U.S. Pat. No.        3,189,526.

Information on the taxonomic classification of strains of this group ofbacteria can be found, inter alia, in Seiler (Journal of GeneralMicrobiology 129, 1433-1477 (1983)), Kinoshita (1985, Glutamic AcidBacteria, p115-142. In: Demain and Solomon (ed), Biology of IndustrialMicroorganisms. The Benjamin/Cummins Publishing Co., London, UK),Kämpfer and Kroppenstedt (Canadian Journal of Microbiology 42, 989-1005(1996)), Liebl et al. (International Journal of Systematic Bacteriology41, 255-260 (1991)) and in U.S. Pat. No. 5,250,434.

Strains denoted “ATCC” may be obtained from the American Type CultureCollection (Manassas, Va., USA). Strains denoted “DSM” may be obtainedfrom the Deutsche Sammlung von Mikroorganismen and Zellkulturen (DSMZ,Brunswick, Germany). Strains denoted “NRRL” may be obtained from theAgricultural Research Service patent Culture Collection (ARS, Peoria,Ill., US). Strains denoted “FERM” may be obtained from the NationalInstitute of Advanced Industrial Science and Technology (AIST TsukubaCentral 6, 1-1-1 Higashi, Tsukuba Ibaraki, Japan).

A gene is chemically a polynucleotide. Another term for this is nucleicacid. The polypeptide with GTP-pyrophosphate kinase activity, encoded bythe rel gene, is also referred to in the prior art as “GTPdiphosphokinase”, “guanosine 3′,5′-polyphosphate synthase” or “stringentfactor” (see, for example, KEGG database (Kyoto Encyclopedia of Genesand Genomes) of Kanehisa Laboratory, Bioinformatics Center, Institutefor Chemical Research, Kyoto University, Japan).

Its EC number is 2.7.6.5. according to the IUPAC (International Union ofPure and Applied Chemistry) nomenclature.

It catalyzes the following reaction:ATP+GTP^(→)AMP+Guanosine 3′-diphosphate 5′-triphosphate,with GDP alsobeing used as substrate instead of GTP.

The term “proteinogenic amino acids” means the amino acids occurring innatural proteins, i.e. in proteins of microorganisms, plants, animalsand humans. However, in the context of the present invention, the term“proteinogenic amino acids” means the group of L-amino acids consistingof L-aspartic acid, L-asparagine, L-threonine, L-serine, L-glutamicacid, L-glutamine, glycine, L-alanine, L-cysteine, L-valine,L-methionine, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine,L-histidine, L-lysine, L-tryptophan, L-proline and L-arginine. L-Aminoacids also include L-homoserine.

The mutants of the invention preferably secrete said proteinogenic aminoacids, more preferably L-lysine, L-tryptophan, L-proline, L-valine,L-isoleucine or L-homoserine. The term “amino acids” also comprisestheir salts such as, for example, lysine monohydrochloride or lysinesulfate in the case of the amino acid L-lysine.

The invention further relates to mutants of coryneform bacteria, whichcomprise a rel allele encoding a polypeptide having a GTP-pyrophosphatekinase enzyme activity, which polypeptide comprises the amino acidsequence of SEQ ID NO: 2, with one of the aforementioned proteinogenicamino acids other than L-proline being present in position 38,preferably L-proline is replaced with L-leucine.

The invention furthermore relates to mutants of coryneform bacteria,which comprise a rel allele encoding a polypeptide havingGTP-pyrophosphate kinase enzyme activity, which polypeptide comprisesone of the proteinogenic amino acids other than L-proline, preferably,L-leucine, in the position corresponding to position 38 of the aminoacid sequence of SEQ ID NO:2, the gene comprising a nucleotide sequenceidentical to the nucleotide sequence of a polynucleotide which isobtainable by a polymerase chain reaction (PCR) using a primer pairwhose nucleotide sequences comprise in each case at least 15 contiguousnucleotides selected from the nucleotide sequence between positions 1and 750 of SEQ ID NO:3 or SEQ ID NO:7 and from the complementarynucleotide sequence between positions 3800 and 3031 of SEQ ID NO:3 orSEQ ID NO:7. One example of a suitable primer pair is depicted in SEQ IDNO:19 and SEQ ID NO:20. The preferred starting material (template DNA)is chromosomal DNA of coryneform bacteria which have been treated inparticular with a mutagen, preferably, the chromosomal DNA of the genusCorynebacterium, and more preferably, the species Corynebacteriumglutamicum.

The invention furthermore relates to mutants of coryneform bacteria,which comprise a rel allele encoding a polypeptide havingGTP-pyrophosphate kinase enzyme activity, which polypeptide comprises anamino acid sequence having a length corresponding to 760 L-amino acids,with one of the proteinogenic amino acids other than L-proline,preferably L-leucine, being present in position 38.

The invention furthermore relates to mutants of coryneform bacteria,which comprise a rel allele encoding a polypeptide havingGTP-pyrophosphate kinase enzyme activity, which polypeptide comprisesthe amino acid sequence corresponding to positions 19 to 57 of SEQ IDNO:6 or 8 in positions 19 to 57 of the amino acid sequence. Preferably,the amino acid sequence of the encoded polypeptide comprises an aminoacid sequence corresponding to positions 9 to 107 of SEQ ID NO:6 or 8 orto positions 9 to 207 of SEQ ID NO:6 or 8 or to positions 9 to 407 ofSEQ ID NO:6 or 8 or to positions 9 to 607 of SEQ ID NO:6 or 8 or topositions 9 to 707 of SEQ ID NO:6 or 8 or to positions 2 to 707 of SEQID NO:6 or 8 or to positions 9 to 757 of SEQ ID NO:6 or 8 or topositions 2 to 757 of SEQ ID NO:6 or 8 or to positions 2 to 758 of SEQID NO:6 or 8 or to positions 2 to 759 of SEQ ID NO:6 or 8, L-glutamicacid being optionally present in position 262, preferably the encodedpolypeptide comprises 760 amino acids.

The invention furthermore relates to mutants of coryneform bacteria,which comprise a rel allele encoding a polypeptide havingGTP-pyrophosphate kinase enzyme activity, which polypeptide comprisesone of the proteinogenic amino acids other than L-proline in position 38or in the corresponding position of the amino acid sequence, preferencebeing given to the substitution with L-leucine, and whose amino acidsequence is moreover at least 90%, preferably at least 92% or at least94% or at least 96%, and more preferably at least 97% or at least 98% orat least 99%, identical to the amino acid sequence of SEQ ID NO:6 or SEQID NO:8. L-Glutamic acid is optionally contained in position 262 of SEQID NO:6 or SEQ ID NO:8.

The invention furthermore relates to mutants of coryneform bacteria,which comprise a rel allele encoding a polypeptide havingGTP-pyrophosphate kinase enzyme activity, which polypeptide comprisesone of the proteinogenic amino acids other than L-proline in position 38or in the corresponding position of the amino acid sequence, withpreference being given to the substitution with L-leucine, and whosenucleotide sequence is moreover at least 90%, preferably at least 92% orat least 94% or at least 96%, and more preferably at least 97% or atleast 98% or at least 99%, identical to the nucleotide sequence of SEQID NO:5. Adenine is optionally present in position 785.

Conservative amino acid substitutions are known to alter the enzymeactivity only insignificantly. Accordingly, the rel allele which ispresent in the mutants of the invention and which encodes a polypeptidehaving GTP-pyrophosphate kinase enzyme activity may comprise one (1) ormore conservative amino acid substitution(s), in addition to the aminoacid sequence depicted in SEQ ID NO:6 and SEQ ID NO:8, preferably, thepolypeptide comprising no more than two (2), no more than three (3), nomore than four (4) or no more than five (5), conservative amino acidsubstitutions. L-Glutamic acid is optionally present in position 262 ofSEQ ID NO:6 or SEQ ID NO:8.

In the case of the aromatic amino acids, the substitutions are said tobe conservative when phenylalanine, tryptophan and tyrosine aresubstituted for one another. In the case of the hydrophobic amino acids,the substitutions are said to be conservative when leucine, isoleucineand valine are substituted for one another. In the case of the polaramino acids, the substitutions are said to be conservative whenglutamine and asparagine are substituted for one another. In the case ofthe basic amino acids, the substitutions are said to be conservativewhen arginine, lysine and histidine are substituted for one another. Inthe case of the acidic amino acids, the substitutions are said to beconservative when aspartic acid and glutamic acid are substituted forone another. In the case of the hydroxyl group-containing amino acids,the substitutions are said to be conservative when serine and threonineare substituted for one another.

During work on the present invention, comparison of the amino acidsequence using the Clustal program (Thompson et al., Nucleic AcidsResearch 22, 4637-4680 (1994)) revealed that the amino acid sequences ofGTP-pyrophosphate kinase of various bacteria such as, for example,Escherichia coli, Bacillus subtilis, Mycobacterium tuberculosis,Mycobacterium bovis, Streptomyces coeliclor, Streptomyces avermitilis,Corynebacterium efficiens and Corynebacterium glutamicum, comprise asequence motif consisting of the sequenceSer-Gly-Asp/Glu-Pro-Tyr-Ile-Thr/Ile-His-Pro-Leu-Ala-Val, a sequencemotif consisting of the sequenceAla-Ala/Gly-Leu-Leu-His-Asp-Thr-Val-Glu-Asp-Thr, and also a sequencemotif consisting of the sequenceThr-Pro-Val-Asp-Phe-Ala-Tyr-Ala-Val-His-Thr-Glu-Val-Gly-His-Arg. Theterms “Asp/Glu”, “Thr/Ile” and “Ala/Gly” mean that “Asp or Glu” or “Thror Ile” or “Ala or Gly” are present in the corresponding position.

Accordingly, those mutants of coryneform bacteria, which comprise a relallele encoding a polypeptide having GTP-pyrophosphate kinase enzymeactivity, which polypeptide comprises at least one amino acid sequenceselected from the group consisting ofSer-Gly-Asp/Glu-Pro-Tyr-Ile-Thr/Ile-His-Pro-Leu-Ala-Val,Ala-Ala/Gly-Leu-Leu-His-Asp-Thr-Val-Glu-Asp-Thr andThr-Pro-Val-Asp-Phe-Ala-Tyr-Ala-Val-His-Thr-Glu-Val-Gly-His-Arg andwhich comprises one of the proteinogenic amino acids other thanL-proline, preferably L-leucine, in position 38 or in the correspondingor comparable position of the amino acid sequence are preferred.

The amino acid sequence motif Ser-Gly-Asp/Glu-Pro-Tyr-Ile-Thr (SEQ IDNO:24)/Ile-His-Pro-Leu-Ala-Val (SEQ ID NO:25) is present, for example,in SEQ ID NO:2 or 4, respectively, or 6 or 8, respectively, frompositions 73 to 84. The amino acid sequence motifAla-Ala/Gly-Leu-Leu-His-Asp-Thr-Val-Glu-Asp-Thr (SEQ ID NO:26) ispresent, for example, in SEQ ID NO:2 or 4, respectively, or 6 or 8,respectively, from position 100 to 110. The amino acid sequence motifThr-Pro-Val-Asp-Phe-Ala-Tyr-Ala-Val-His-Thr-Glu-Val-Gly-His-Arg (SEQ IDNO:27) is present, for example, in SEQ ID NO:2 or 4, respectively, or 6or 8, respectively, from positions 437 to 452.

The invention relates to mutants of coryneform bacteria, which comprisea rel allele encoding a polypeptide having GTP-pyrophosphate kinaseenzyme activity, which polypeptide comprises the amino acid sequence ofSEQ ID NO:6 or SEQ ID NO:8, respectively, L-glutamic acid beingoptionally present in position 262.

Enzymes intrinsic to the host, called aminopeptidases, are known toremove the terminal methionine during protein synthesis.

The term “a position corresponding to position 38 of the amino acidsequence” or “a position comparable to position 38 of the amino acidsequence” means that an insertion or deletion of a codon coding for anamino acid in the N-terminal region (based on position 38 of SEQ ID NO:6or 8) of the encoded polypeptide formally increases, in the case of aninsertion, or decreases, in the case of a deletion, the indicatedposition and indicated length, in each case by one unit. For example,deletion of the GAG codon coding for the amino acid L-glutamic acid inposition 4 of SEQ ID NO:6 or 8 moves the L-leucine from position 38 toposition 37. The indicated length would then be: 759 amino acids. In thesame way, insertion or deletion of a codon coding for an amino acid inthe C-terminal region (based on position 38) of the encoded polypeptideformally increases, in the case of an insertion, or decreases, in thecase of a deletion, the indicated length by one unit. Such comparablepositions can readily be identified by comparing the amino acidsequences in the form of an alignment, for example, with the aid of theClustal program or the MAFFT program.

Insertions and deletions of this kind essentially do not affect theenzymic activity. “Essentially do not affect” means that the enzymicactivity of the variants mentioned differs from the activity of thepolypeptide having the amino acid sequence of SEQ ID NO:6 or 8,respectively, by no more than 10%, no more than 7.5%, no more than 5%,no more than 2.5% or no more than 1%, L-glutamic acid being optionallypresent in position 262.

Accordingly, the invention also relates to rel alleles encodingpolypeptide variants of SEQ ID NO:6 or 8, respectively, which variantscomprise one or more insertion(s) or deletion(s), L-glutamic acid beingoptionally present in position 262. The polypeptide preferably comprisesno more than 5, no more than 4, no more than 3 or no more than 2 aminoacid insertions or deletions.

The abovementioned sequence motifs Ser-Gly-Asp/Glu-Pro-Tyr-Ile-Thr (SEQID NO:24)/Ile-His-Pro-Leu-Ala-Val (SEQ ID NO:25),Ala-Ala/Gly-Leu-Leu-His-Asp-Thr-Val-Glu-Asp-Thr (SEQ ID NO:26) andThr-Pro-Val-Asp-Phe-Ala-Tyr-Ala-Val-His-Thr-Glu-Val-Gly-His-Arg (SEQ IDNO:27) are preferably not disrupted by such insertions/deletions.

The mutants of the invention may be prepared by classical in-vivomutagenesis methods with cell populations of coryneform bacteria byusing mutagenic substances such as, for example,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate(EMS), 5-bromouracil, or ultraviolet light. Mutagenesis methods aredescribed, for example, in Manual of Methods for General Bacteriology(Gerhard et al. (Eds.), American Society for Microbiology, Washington,D.C., USA, 1981) or in Tosaka et al. (Agricultural and BiologicalChemistry 42(4), 745-752 (1978)) or in Konicek et al. (FoliaMicrobiologica 33, 337-343 (1988)). Typical mutageneses using MNNGcomprise concentrations of from 50 to 500 mg/l or else higherconcentrations of up to a maximum of 1 g/l, an incubation time of from 1to 30 minutes at a pH of from 5.5 to 7.5. Under these conditions, thenumber of viable cells is reduced by a proportion of from approx. 50% to90% or approx. 50% to 99% or approx. 50% to 99.9% or more.

Mutants or cells are removed from the mutagenized cell population andpropagated. In a further step, the ability of the mutants or cells tosecrete amino acids, preferably L-lysine, L-tryptophan, L-proline,L-valine, L-isoleucine or L-homoserine in a batch culture using asuitable nutrient medium is investigated. Suitable nutrient media andassay conditions are described, inter alia, in U.S. Pat. No. 6,221,636,in U.S. Pat. No. 5,840,551, in U.S. Pat. No. 5,770,409, in U.S. Pat. No.5,605,818, in U.S. Pat. No. 5,275,940 and in U.S. Pat. No. 4,224,409.When suitable robots are used, such as, for example, in Zimmermann etal. (VDI Berichte No. 1841, VDI-Verlag, Dusseldorf, Germany 2004,439-443) or Zimmermann (Chemie Ingenenieur Technik 77 (4), 426-428(2005)), it is possible to study numerous mutants within a short periodof time. Usually no more than 3000, no more than 10 000, no more than 30000 or else no more than 60 000, where appropriate also more, mutantsare studied. In this way, mutants are identified which, compared to theparent strain or non-mutagenized starting strain, secrete an increasedamount of amino acids into the nutrient medium or the cell interior.These include, for example, those mutants whose amino acid secretion hasincreased by at least 0.5%.

Subsequently, DNA of the mutants is provided or isolated from the latterand the corresponding polynucleotide is synthesized with the aid of thepolymerase chain reaction using primer pairs which allow amplificationof the rel gene or of the rel allele of the invention or of the mutationof the invention in position 38. Preferably, the DNA from those mutantswhich secrete an increased amount of amino acids is isolated.

It is possible to select any primer pairs from the nucleotide sequencelocated upstream and downstream of the mutation of the invention andfrom the nucleotide sequence complementary thereto. A primer of a primerpair here preferably comprises at least 15, at least 18, at least 20, atleast 21 or at least 24, contiguous nucleotides selected from thenucleotide sequence between positions 1 and 861 of SEQ ID NO:3 or SEQ IDNO:7. The corresponding second primer of a primer pair comprises atleast 15, at least 18, at least 20, at least 21 or at least 24,contiguous nucleotides selected from the complementary nucleotidesequence of positions 3800 and 865 of SEQ ID NO:3 or SEQ ID NO:7. If itis desired to amplify the coding region, then the primer pair ispreferably selected from the nucleotide sequence between positions 1 and750 of SEQ ID NO:3 or SEQ ID NO:7 and from the complementary nucleotidesequence between positions 3800 and 3031 of SEQ ID NO:3 or SEQ ID NO:7.

If it is desired to amplify part of the coding region, as indicated, forexample, in SEQ ID NO:11 and 13, then the primer pair is preferablyselected from the nucleotide sequence between positions 751 and 804 orbetween positions 1 and 804 of SEQ ID NO:3 or SEQ ID NO:7 and from thecomplementary nucleotide sequence between positions 3030 and 922 or 3800and 922 of SEQ ID NO:3 or SEQ ID NO:7. An example of a suitable primerpair is the rel_XL_A1 and rel_X_E1 primer pair depicted under SEQ IDNO:19 and SEQ ID NO:20. In addition, the primer may be provided withrecognition sites for restriction enzymes, with a biotin group orfurther accessories as described in the prior art. The total length ofthe primer is no more than 30, 40, 50 or 60 nucleotides.

Thermostable DNA polymerases are employed in the preparation ofpolynucleotides by amplification of selected sequences such as the relallele of the invention from initially introduced DNA, for examplechromosomal DNA (template DNA), via amplification by means of PCR.Examples of DNA polymerases of this kind are Taq polymerase of Thermusaquaticus, which is sold, inter alia, by Qiagen (Hilden, Germany), Ventpolymerase of Thermococcus litoralis, sold, inter alia, by New EnglandBiolabs (Frankfurt, Germany), or Pfu polymerase of Pyrococcus furiosus,sold, inter alia, by Stratagene (La Jolla, USA). Preference is given topolymerases having proof-reading activity. Proof-reading activity meansthat these polymerases are capable of recognizing wrongly incorporatednucleotides and rectifying the error by renewed polymerization(Lottspeich and Zorbas, Bioanalytik, Spektrum Akademischer Verlag,Heidelberg, Germany (1998)). Examples of polymerases havingproof-reading activity are Vent polymerase and Pfu polymerase.

The conditions in the reaction mixture are set according to theinformation provided by the manufacturer. The polymerases are usuallysupplied by the manufacturer together with the customary buffer whichusually has concentrations of 10-100 mM Tris/HCl and 6-55 mM KCl at pH7.5-9.3. Magnesium chloride is added in a concentration of 0.5-10 mM, ifnot present in the buffer supplied by the manufacturer. Furthermore,deoxynucleoside triphosphates are added in a concentration of 0.1-16.6mM to the reaction mixture. The primers, in a final concentration of0.1-3 μM, and template DNA, in the optimal case from 10² to 10⁵ copies,are initially introduced into the reaction mixture. 10⁶ to 10⁷ copiesmay also be used. An amount of 2-5 units of the appropriate polymeraseis added to the reaction mixture. A typical reaction mixture has avolume of 20-100 μl.

Further additives which may be added to the reaction are bovine serumalbumin, Tween-20, gelatin, glycerol, formamide or DMSO (Dieffenbach andDveksler, PCR Primer—A Laboratory Manual, Cold Spring Harbor LaboratoryPress, USA 1995).

A typical PCR profile consists of three different, successively repeatedtemperature stages. Initially, the reaction is started by increasing thetemperature to 92° C.-98° C. for 4 to 10 minutes in order to denaturethe initially introduced DNA. This is followed repeatedly by first astep of denaturing the initially introduced DNA at approximately 92-98°C. for 10-60 seconds, then a step of 10-60 seconds of binding theprimers to the initially introduced DNA at a particular temperaturedependent on said primers (annealing temperature), which from experienceis from 50° C. to 60° C. and can be calculated for each primer pairindividually. Detailed information on this can be found by the skilledworker in Rychlik et al. (Nucleic Acids Research 18 (21): 6409-6412).Subsequently, a synthesis step of extending the initially introducedprimers (extension) at the activity optimum of the polymerase, indicatedin each case and usually in the range from 73° C. to 67° C., preferably72° C. to 68° C., depending on the polymerase. The duration of thisextension step depends on the performance of the polymerase and on thelength of the PCR product to be amplified. In a typical PCR, this steplasts 0.5-8 minutes, preferably 2-4 minutes. These three steps arerepeated 30 to 35 times, where appropriate up to 50 times. A final“extension” step of 4-10 minutes ends the reaction. The polynucleotidesprepared in this manner are also referred to as amplicons; the termnucleic acid fragment is likewise common.

Further instructions and information regarding PCR can be found by theskilled worker, for example, in the manual “PCR-Strategies” (Innis,Felfand and Sninsky, Academic Press, Inc., 1995), in the manual byDiefenbach and Dveksler “PCR Primer—a laboratory manual” (Cold SpringHarbor Laboratory Press, 1995), in the manual by Gait “Oligonucleotidesynthesis: A Practical Approach” (IRL Press, Oxford, UK, 1984) and inNewton and Graham “PCR” (Spektrum Akademischer Verlag, Heidelberg,Germany, 1994).

The nucleotide sequence is subsequently determined, for example by thechain termination method of Sanger et al. (Proceedings of the NationalAcademies of Sciences, U.S.A., 74, 5463-5467 (1977)) with themodifications indicated by Zimmermann et al. (Nucleic Acids Research 18,1067pp (1990)), and the polypeptide encoded by said nucleotide sequenceis analyzed, in particular with respect to the amino acid sequence. Forthis purpose, the nucleotide sequence is entered into a program fortranslating DNA sequence into an amino acid sequence. Examples ofsuitable programs are the program “Patentin” which is available frompatent offices, for example the US Patent and Trademark Office (USPTO),or “Translate Tool” which is available on the ExPASy Proteomics Serveron the World Wide Web (Gasteiger et al., Nucleic Acids Research 31,3784-3788 (2003)).

In this way, mutants are identified whose rel alleles encodepolypeptides having GTP-pyrophosphate kinase enzyme activity, whichpolypeptides comprise one of the proteinogenic amino acids other thanL-proline in position 38 of the amino acid sequence or in thecorresponding or comparable position, preferably, the substitution withL-leucine.

Where appropriate, the entire chromosome of the mutant is determined.This may involve using the method described by margulies et al. (Nature,437(7057): 376-380 (2005) and Velicer et al. (Proceedings of theNational Academy of Sciences, USA., 103(21), 8107-8112 (2006)), which isknown under the keyword “pyro-sequencing” in the art and enablescomplete genomes to be sequenced rapidly.

Accordingly, the invention relates to a mutant of a coryneformbacterium, which is obtainable by a method comprising:

-   -   a) treating a coryneform bacterium capable of secreting amino        acids with a mutagenic agent,    -   b) isolating and propagating the mutant generated in a),    -   c) preferably determining the ability of said mutant to secrete        in a medium or to accumulate in the cell interior at least 0.5%        more amino acid than the coryneform bacterium employed in a),    -   d) providing nucleic acid of the mutant obtained in b),    -   e) preparing a nucleic acid molecule (amplicon or nucleic acid        fragment, respectively) using the polymerase chain reaction, of        the nucleic acid from d) and of a primer pair consisting of a        first primer comprising at least 15 contiguous nucleotides        selected from the nucleotide sequence between positions 1 and        861, preferably 1, and 750 of SEQ ID NO:3 or SEQ ID NO:7 and a        second primer comprising at least 15 contiguous nucleotides        selected from the complementary nucleotide sequence between        positions 3800 and 865, preferably 3800, and 3031 of SEQ ID NO:3        or 7,    -   f) determining the nucleotide sequence of the nucleic acid        molecule obtained in e) and determining the encoded amino acid        sequence,    -   g) comparing, where appropriate, the amino acid sequence        determined in f) with SEQ ID NO:6 or 8 L-glutamic acid being        optionally present in position 262, and    -   h) identifying a mutant comprising a polynucleotide which        encodes a polypeptide comprising one of the proteinogenic amino        acids other than L-proline, preferably L-leucine, in position 38        or a comparable position.

The mutants generated in this way typically comprise one (1) copy of therel allele described.

SEQ ID NO:5 depicts, by way of example, the coding region of rel alleleof a mutant of the invention. The coding region of the wild type gene isdepicted as SEQ ID NO:1. SEQ ID NO:1 comprises the nucleobase cytosinein position 112, the nucleobase cytosine in position 113 and thenucleobase guanine in position 114. SEQ ID NO:1 thus comprises the CCGcodon, coding for the amino acid L-proline, from positions 112 to 114.SEQ ID NO:5 comprises the nucleobase thymine in position 113. Thiscytosine-thymine transition results in the CTG codon, coding for theamino acid L-leucine, in positions 112 to 114.

In addition, the nucleotide sequences depicted in SEQ ID NO: 5 and 7,respectively, may comprise further base substitutions which haveresulted from the mutagenesis treatment but which do not manifestthemselves in an altered amino acid sequence. Such mutations arereferred to in the art also as silent or neutral mutations. These silentmutations may likewise already be present in the coryneform bacteriumused for the mutagenesis treatment.

The coryneform bacteria used for the mutagenesis preferably already havethe ability to secrete the desired amino acid into the surroundingnutrient medium or fermentation broth or to accumulate it in the cellinterior.

L-Lysine-producing coryneform bacteria typically possess afeedback-resistant or desensibilized aspartate kinase.Feedback-resistant aspartate kinases mean aspartate kinases (LysC)which, compared to the wild type, have a lower sensitivity to theinhibition by mixtures of lysine and threonine or mixtures of AEC(aminoethylcysteine) and threonine or lysine alone or AEC alone. Thegenes or alleles encoding these desensibilized aspartate kinases arealso referred to as lysC^(FBR) alleles. The prior art describes numerouslysC^(FBR) alleles encoding aspartate kinase variants which have aminoacid substitutions in comparison with the wild type protein. SEQ ID NO:9depicts the coding region of the wild type lysC gene of Corynebacteriumglutamicum according to accession number AX756575 of the NCBI database,and SEQ ID NO:10 depicts the polypeptide encoded by said gene.

The L-lysine-producing coryneform bacteria employed for the purposes ofthe invention have preferably an lysC allele encoding an aspartatekinase variant whose amino acid sequence is that of SEQ ID NO:10comprising one or more of the amino acid substitutions selected from thegroup consisting of:

LysC A279T (replacing L-alanine in position 279 of the encoded aspartatekinase protein according to sequence SEQ ID NO:10 with L-threonine; seeU.S. Pat. No. 5,688,671 and Accession numbers E06825, E06826, E08178 andI74588 to I74597),

LysC A279V (replacing L-alanine in position 279 of the encoded aspartatekinase protein according to sequence SEQ ID NO:10 with L-valine, see JP6-261766 and Accession number E08179),

LysC L297Q (replacing L-leucine in position 297 of the encoded aspartatekinase protein according to sequence SEQ ID NO:10 with L-glutamine; seeDE 102006026328,

LysC S301F (replacing L-serine in position 301 of the encoded aspartatekinase protein according to sequence SEQ ID NO:10 with L-phenylalanine;see U.S. Pat. No. 6,844,176 and Accession number E08180),

LysC S301Y (replacing L-serine in position 301 of the encoded aspartatekinase protein according to sequence SEQ ID NO:10 with L-tyrosine, seeKalinowski et al. (Molecular and General Genetics 224, 317-324 (1990))and Accession number X57226),

LysC T308I (replacing L-threonine in position 308 of the encodedaspartate kinase protein according to sequence SEQ ID NO:10 withL-isoleucine; see JP 6-261766 and Accession number E08181).

LysC T311I (replacing L-threonine in position 311 of the encodedaspartate kinase protein according to sequence SEQ ID NO:10 withL-isoleucine; see WO 00/63388 and U.S. Pat. No. 6,893,848),

LysC S317A (replacing L-serine in position 317 of the encoded aspartatekinase protein according to sequence SEQ ID NO:10 with L-alanine; seeU.S. Pat. No. 5,688,671 and Accession number I74589),

LysC R320G (replacing L-arginine in position 320 of the encodedaspartate kinase protein according to sequence SEQ ID NO:10 withglycine; see Jetten et al. (Applied Microbiology and Biotechnology 43,76-82 (995)) and Accession number L27125),

LysC G345D (replacing glycine in position 345 of the encoded aspartatekinase protein according to sequence SEQ ID NO:10 with L-aspartic acid;see Jetten et al. (Applied Microbiology and Biotechnology 43, 76-82(995)) and Accession number L16848),

LysC T380I (replacing L-threonine in position 380 of the encodedaspartate kinase protein according to sequence SEQ ID NO:10 withL-isoleucine; see WO 01/49854 and Accession number AX192358), and

LysC S381F (replacing L-serine in position 381 of the encoded aspartatekinase protein according to sequence SEQ ID NO:10 with L-phenylalanine;see EP 0435132).

The lysC^(FBR) allele, lysC T311I (replacing threonine in position 311of the encoded aspartate kinase protein according to SEQ ID NO:10 withisoleucine) and a lysC^(FBR) allele comprising at least one substitutionselected from the group consisting of A279T (replacing alanine inposition 279 of the encoded aspartate kinase proteins according to SEQID NO:10 with threonine), S381F (replacing serine in position 381 of theencoded aspartate kinase protein according to SEQ ID NO:10 with phenylalanine) and S317A (replacing serine in position 317 of the encodedaspartate kinase protein according to SEQ ID NO:10 with alanine) arepreferred. More preferable is the lysC^(FBR) allele lysC T311I(replacing threonine in position 311 of the encoded aspartate kinaseprotein according to SEQ ID NO:10 with isoleucine).

The strain DSM 16833 (WO 06/063660) has a lysC^(FBR) allele whichencodes an aspartate kinase protein comprising the amino acidsubstitution T311I.

The strain NRRL B-11474 (U.S. Pat. No. 4,275,157) has a lysC^(FBR)allele which encodes an aspartate kinase protein comprising the aminoacid substitutions A279T and S381F.

Starting from strain DSM 16833, a mutant referred to as DM1915, whichharbors a rel allele encoding a polypeptide in which L-leucine ispresent in position 38 of the amino acid sequence, was isolated in themanner described above. The nucleotide sequence of the coding region ofthe rel allele of the DM1915 mutant is depicted as SEQ ID NO:5 and theamino acid sequence of the encoded polypeptide is depicted as SEQ IDNO:6 and 8, respectively.

In addition, it is possible to use L-lysine-secreting coryneformbacteria which possess properties as known from the prior art.

L-Tryptophan-producing coryneform bacteria typically possess afeedback-resistant or desensibilized anthranilate synthase. The termfeedback-resistant anthranilate synthase (TrpE) means anthranilatesynthases which, compared to the wild type, have a lower sensitivity ofat least 5% to 10%, or at least 10% to 15% or at least 10% to 20% toinhibition by tryptophan or 5-fluorotryptophan (Matsui et al., Journalof Bacteriology 169 (11): 5330-5332 (1987)) or similar analogs. Thegenes or alleles encoding these desensibilized anthranilate synthasesare also referred to as trpE^(FBR) alleles.

Examples of mutants or alleles of this kind are described, for example,in U.S. Pat. No. 6,180,373 and EP0338474.

L-Proline-producing coryneform bacteria have inter alia a γ-glutamylkinase (ProB) which has a proteinogenic amino acid other than glycine inamino acid position 149 or a comparable position, preferably L-asparticacid (WO06066758).

L-Valine-producing coryneform bacteria typically have a“feedback”-resistant or desensitized acetolactate synthase (acetohydroxyacid synthase; EC No. 2.2.1.6).

“Feedback”-resistant acetolactate synthase means an acetolactatesynthase which, in comparison with the wild type, has lower sensitivityto inhibition by one or more of the amino acids selected from the groupconsisting of L-valine, L-isoleucine and L-leucine, preferably L-valine.

The acetolactate synthase (IlvB, IlvN) of Corynebacterium consists of a“large” subunit encoded by the ilvB gene and a “small” subunit encodedby the ilvN gene (Keilhauer et al., Journal of Bacteriology 175(17),5595-5603 (1993)). WO 05/003357 and Elisakova et al. (Applied andEnvironmental Microbiology 71(1):207-13 (2005)) report on variants ofthe IlvN subunit which convey the acetolactate synthase resistance toL-valine, L-isoleucine and L-leucine. The amino acid sequence of onevariant comprises L-aspartic acid instead of L-isoleucine in position 21(IlvN I21D) and L-phenylalanine instead of L-isoleucine in position 22(IlvN I22F). The amino acid sequence of the second variant comprisesL-aspartic acid instead of glycine in position 20 (IlvN G20D),L-aspartic acid instead of L-isoleucine in position 21 (IlvN I21D) andL-phenylalanine instead of L-isoleucine in position 22 (IlvN I22F).

L-Isoleucine-producing coryneform bacteria typically have a“feedback”-resistant or -desensitized threonine dehydratase (=threoninedeaminase).

“Feedback”-resistant threonine dehydratase means a threonine dehydratase(EC No. 4.3.1.19) which, in comparison with the wild type, has lowersensitivity to inhibition by L-isoleucine. The genes or alleles codingfor this desensitized threonine dehydratase are also referred to asilvA^(FBR) alleles.

SEQ ID NO:17 depicts the coding region of the Corynebacterium glutamicumWildtype ilvA gene according to the Accession numbers L01508 andNC_(—)006958 of the NCBI database and SEQ ID NO:18 depicts thepolypeptide encoded by this gene.

The threonine dehydratase variants described in U.S. Pat. No. 6,107,063and in Morback et al. (Applied and Environmental Microbiology 61 (12),4315-4320 (1995)) comprise one or more of the amino acid substitutionsselected from the group consisting of:

-   -   IlvA M199V (replacing L-methionine in position 199 of the        encoded threonine dehydratase protein according to SEQ ID NO:18        with L-valine; see U.S. Pat. No. 6,107,063),    -   IlvA A257G (replacing L-alanine in position 257 of the encoded        threonine dehydratase protein according to SEQ ID NO:18 with        L-arginine; see U.S. Pat. No. 6,107,063),    -   IlvA H278R (replacing L-histidine in position 278 of the encoded        threonine dehydratase protein according to SEQ ID NO:18 with        L-arginine; see U.S. Pat. No. 6,107,063),    -   IlvA V323A (replacing L-valine in position 323 of the encoded        threonine dehydratase protein according to SEQ ID NO:18 with        L-alanine; see Morbach et al.),    -   IlvA L351S (replacing L-leucine in position 351 of the encoded        threonine dehydratase protein according to SEQ ID NO:18 with        L-serine; see U.S. Pat. No. 6,107,063),    -   IlvA D378G (replacing L-aspartic acid in position 378 of the        encoded threonine dehydratase protein according to SEQ ID NO:18        with glycine; see Morbach et al.),

The mutants obtained show increased secretion or production of thedesired amino acid in a fermentation process, in comparison with thestarting strain or parent strain employed.

The invention likewise relates to an isolated polynucleotide encoding apolypeptide having GTP-pyrophosphate kinase enzyme activity, whichpolypeptide comprises one of the proteinogenic amino acids other thanL-proline in position 38 or in a corresponding or comparable position ofthe amino acid sequence, with preference being given to the substitutionwith L-leucine.

The polynucleotide of the invention may be isolated from a mutant of theinvention.

It is furthermore possible to use in-vitro methods for the mutagenesisof the rel gene. The use of in-vitro methods involves subjectingisolated polynucleotides which comprise a rel gene of a coryneformbacterium, preferably the Corynebacterium glutamicum wild type genedescribed in the prior art, to a mutagenic treatment.

The isolated polynucleotides may be, for example, isolated total DNA orchromosomal DNA or else amplicons of the rel gene, which have beenprepared with the aid of the polymerase chain reaction (PCR). Suchamplicons are also referred to as PCR products. Instructions for theamplification of DNA sequences with the aid of the polymerase chainreaction can be found by the skilled worker, inter alia, in the manualby Gait: Oligonucleotide Synthesis: A Practical Approach (IRL Press,Oxford, UK, 1984) and Newton and Graham: PCR (Spektrum AkademischerVerlag, Heidelberg, Germany, 1994). It is likewise possible toincorporate the rel gene to be mutagenized first into a vector, forexample into a bacteriophage or into a plasmid.

Suitable methods of in-vitro mutagenesis are, inter alia, the treatmentwith hydroxylamine according to Miller (Miller, J. H.: A Short Course inBacterial Genetics. A Laboratory Manual and Handbook for Escherichiacoli and Related Bacteria, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, 1992), the use of mutagenic oligonucleotides (T. A.Brown: Gentechnologie für Einsteiger [Genetic engineering forbeginners], Spektrum Akademischer Verlag, Heidelberg, 1993 and R. M.Horton: PCR-Mediated Recombination and Mutagenesis, MolecularBiotechnology 3, 93-99 (1995)) and the use of a polymerase chainreaction using a DNA polymerase with a high error rate. An example ofsuch a DNA polymerase is the Mutazyme DNA Polymerase (GeneMorph PCRMutagenesis Kit, No. 600550) from Stratagene (La Jolla, Calif., USA).

Further instructions and reviews on the generation of mutations in vivoor in vitro can be found in the prior art and in known textbooks ofgenetics and molecular biology, such as, for example, the textbook byKnippers (“Molekulare Genetik”, 6th edition, Georg Thieme Verlag,Stuttgart, Germany, 1995), that by Winnacker (“Gene and Klone”, VCHVerlagsgesellschaft, Weinheim, Germany, 1990) or that by Hagemann(“Allgemeine Genetik”, Gustav Fischer Verlag, Stuttgart, 1986).

The invention furthermore relates to an isolated polynucleotide encodinga polypeptide having GTP-pyrophosphate kinase enzyme activity, whichpolypeptide comprises the amino acid sequence of SEQ ID NO:2, with oneof the proteinogenic amino acids other than L-proline being present inposition 38 of said amino acid sequence, preferably the substitutionwith L-leucine. L-Glutamic acid is optionally contained in position 262of SEQ ID NO:2.

The invention furthermore relates to an isolated polynucleotide encodinga polypeptide having GTP-pyrophosphate kinase enzyme activity, whichpolypeptide comprises an amino acid sequence having a length of 760amino acids, with one of the proteinogenic L-amino acids other thanL-proline, preferably L-leucine, being present in position 38.

The invention furthermore relates to an isolated polynucleotide encodinga polypeptide having GTP-pyrophosphate kinase enzyme activity, whichpolypeptide comprises, in positions 19 to 57 of the amino acid sequence,the amino acid sequence corresponding to positions 19 to 57 of SEQ IDNO:6 or 8 respectively. The amino acid sequence of the encodedpolypeptide preferably comprises an amino acid sequence corresponding topositions 9 to 107 of SEQ ID NO:6 or 8, respectively, or to positions 9to 207 of SEQ ID NO:6 or 8, respectively, or to positions 9 to 407 ofSEQ ID NO:6 or 8, respectively, or to positions 9 to 607 of SEQ ID NO:6or 8, respectively, or to positions 9 to 707 of SEQ ID NO:6 or 8,respectively, or to positions 2 to 707 of SEQ ID NO:6 or 8,respectively, or to positions 9 to 757 of SEQ ID NO:6 or 8,respectively, or to positions 2 to 757 of SEQ ID NO:6 or 8,respectively, or to positions 2 to 758 of SEQ ID NO:6 or 8,respectively, or to positions 2 to 759 of SEQ ID NO:6 or 8,respectively, L-glutamic acid being optionally present in position 262.The length of the encoded polypeptide comprises very particularlypreferably 760 amino acids.

The invention furthermore relates to an isolated polynucleotide encodinga polypeptide having GTP-pyrophosphate kinase enzyme activity, whichpolypeptide comprises one of the proteinogenic amino acids other thanL-proline, preferably L-leucine, in position 38 of the amino acidsequence or in a corresponding or comparable position, and comprising anucleotide sequence identical to the nucleotide sequence of apolynucleotide which is obtainable by a polymerase chain reaction (PCR)using the primer pair whose nucleotide sequences comprise in each caseat least 15 contiguous nucleotides selected from the nucleotide sequencebetween positions 1 and 861, preferably between positions 1 and 750 ofSEQ ID NO:3 or SEQ ID NO:7 and from the complementary nucleotidesequence between positions 3800 and 865, preferably between positions3800 and 3031 of SEQ ID NO:3 or SEQ ID NO:7. One example of a suitableprimer pair of this kind is depicted in SEQ ID NO:19 and SEQ ID NO:20.The preferred starting material (template DNA) is chromosomal DNA ofcoryneform bacteria, in particular of those which have been treated witha mutagen, preferably, the chromosomal DNA of the genus Corynebacterium,and more preferably, the species Corynebacterium glutamicum.

The invention furthermore relates to an isolated polynucleotide whichhybridizes with the nucleotide sequence complementary to SEQ ID NO:5under stringent conditions and which encodes a polypeptide havingGTP-pyrophosphate kinase enzyme activity, which polypeptide comprisesone of the proteinogenic amino acids other than L-proline, preferablyL-leucine, in position 38 of the amino acid sequence or in acorresponding or comparable position.

Instructions regarding the hybridization of nucleic acids orpolynucleotides can be found by the skilled worker, inter alia, in themanual “The DIG System User's Guide for Filter Hybridization” fromBoehringer Mannheim GmbH (Mannheim, Germany, 1993) and in Liebl et al.(International Journal of Systematic Bacteriology 41: 255-260 (1991)).The hybridization is carried out under stringent conditions, i.e. onlyhybrids in which the probe, i.e. a polynucleotide comprising thenucleotide sequence complementary to SEQ ID NO:5, and the targetsequence, i.e. the polynucleotides treated or identified with the probe,are at least 90% identical, are formed. The stringency of thehybridization, including that of the washing steps, is known to beinfluenced or determined by varying the buffer composition, thetemperature and the salt concentration. The hybridization reaction iscarried out at relatively low stringency compared to the washing steps(Hybaid Hybridisation Guide, Hybaid Limited, Teddington, UK, 1996).

For example, a buffer corresponding to 5×SSC buffer at a temperature ofapprox. 50° C.-68° C. may be used for the hybridization reaction. Inthis case, probes may also hybridize with polynucleotides which are lessthan 90% identical to the nucleotide sequence of the probe employed.Such hybrids are less stable and are removed by washing under stringentconditions. This may be achieved, for example, by lowering the saltconcentration to 2×SSC and, where appropriate, subsequently to 0.5×SSC(The DIG System User's Guide for Filter Hybridisation, BoehringerMannheim, Mannheim, Germany, 1995), with the temperature being set toapprox. 50° C.-68° C., approx. 52° C.-68° C., approx. 54° C.-68° C.,approx. 56° C.-68° C., approx. 58° C.-68° C., approx. 60° C.-68° C.,approx. 62° C.-68° C., approx. 64° C.-68° C., approx. 66° C.-68° C.Temperature ranges of approx. 64° C.-68° C. or approx. 66° C.-68° C. arepreferred. It is possible, where appropriate, to lower the saltconcentration down to a concentration corresponding to 0.2×SSC or0.1×SSC. The SSC buffer comprises, where appropriate, sodium dodecylsulfate (SDS) in a concentration of 0.1%. By gradually increasing thehybridization temperature in steps of approx. 1-2° C. from 50° C. to 68°C., it is possible to isolate polynucleotide fragments which have atleast 90% or at least 91%, preferably at least 92% or at least 93% or atleast 94% or at least 95% or at least 96%, and more preferably at least97% or at least 98% or at least 99%, identity to the sequence orcomplementary sequence of the probe employed and which encode apolypeptide which has GTP-pyrophosphate kinase enzyme activity andcomprises the amino acid substitution of the invention. The nucleotidesequence of the polynucleotide obtained in this way is determined byknown methods. Further instructions regarding hybridization arecommercially available in the form of “kits” (e.g. DIG Easy Hyb fromRoche Diagnostics GmbH, Mannheim, Germany, Catalog No. 1603558). Thenucleotide sequences thus obtained encode polypeptides havingGTP-pyrophosphate kinase enzyme activity, which polypeptides are atleast 90%, preferably at least 92% or at least 94% or at least 96%, andvery particularly preferably at least 97% or at least 98% or at least99%, identical to the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:8,respectively, and which comprise the amino acid substitution of theinvention, L-glutamic acid being optionally present in position 262.

The invention furthermore relates to an isolated polynucleotide encodinga polypeptide having GTP-pyrophosphate kinase enzyme activity, whichpolypeptide comprises one of the proteinogenic amino acids other thanL-proline in position 38 or in a corresponding or comparable position ofthe amino acid sequence, the substitution with L-leucine beingpreferred, and which comprises an amino acid sequence which moreover isat least 90%, preferably at least 92% or at least 94% or at least 96%,and very particularly preferably at least 97% or at least 98% or atleast 99%, identical to the amino acid sequence of SEQ ID NO:6 or SEQ IDNO:8, respectively, L-glutamic acid being optionally present in position262.

The invention furthermore relates to an isolated polynucleotide encodinga polypeptide having GTP-pyrophosphate kinase enzyme activity, whichpolypeptide comprises one of the proteinogenic amino acids other thanL-proline in position 38 or in a corresponding or comparable position ofthe amino acid sequence, the substitution with L-leucine beingpreferred, and comprising a nucleotide sequence which moreover is atleast 90%, preferably at least 92% or at least 94% or at least 96%, andvery particularly preferably at least 97% or at least 98% or at least99%, identical to the nucleotide sequence of SEQ ID NO:5, adenine beingoptionally present in position 785.

In addition, preference is given to those isolated polynucleotidesencoding a polypeptide having GTP-pyrophosphate kinase enzyme activity,which polypeptide comprises one of the proteinogenic amino acids otherthan L-proline, preferably L-leucine, in position 38 of the amino acidsequence or in a corresponding or comparable position, and comprising atleast one sequence motif or an amino acid sequence selected from thegroup consisting of Ser-Gly-Asp/Glu-Pro-Tyr-Ile-Thr (SEQ IDNO:24)/Ile-His-Pro-Leu-Ala-Val (SEQ ID NO:25),Ala-Ala/Gly-Leu-Leu-His-Asp-Thr-Val-Glu-Asp-Thr (SEQ ID NO:26) andThr-Val-Asp-Phe-Ala-Tyr-Ala-Val-His-Thr-Glu-Val-Gly-His-Arg (SEQ IDNO:27).

The terms “Asp/Glu”, “Thr/Ile” and “Ala/Gly” mean that “Asp or Glu” or“Thr or Ile” or “Ala or Gly” are present in the corresponding position.

The invention furthermore relates to an isolated polynucleotide encodinga polypeptide having GTP-pyrophosphate kinase enzyme activity, whichpolypeptide comprises the amino acid sequence of SEQ ID NO:6 or 8,respectively, L-glutamic acid being optionally present in position 262.The encoded polypeptide comprises, where appropriate, one (1) or moreconservative amino acid substitution(s). Preferably, the polypeptidecomprises no more than two (2), no more than three (3), no more thanfour (4) or no more than five (5), conservative amino acidsubstitutions.

The invention furthermore relates to an isolated polynucleotide encodinga polypeptide having GTP-pyrophosphate kinase enzyme activity, whichpolypeptide comprises the amino acid sequence of SEQ ID NO:6 or 8,respectively, including an extension at the N- or C-terminus by at leastone (1) amino acid. This extension has no more than 50, 40, 30, 20, 10,5, 3 or 2 amino acids or amino acid residues. L-Glutamic acid beingoptionally contained in position 262 of SEQ ID NO:6 or 8, respectively.

The invention relates to rel alleles encoding polypeptide variants ofSEQ ID NO:6 or 8, respectively, L-glutamic acid being optionally presentin position 262, which comprise one or more insertions or deletions.These preferably comprise no more than 5, no more than 4, no more than 3or no more than 2 insertions or deletions of amino acids. Preferably,the sequence motifs Ser-Gly-Asp/Glu-Pro-Tyr-Ile-Thr (SEQ IDNO:24)/Ile-His-Pro-Leu-Ala-Val (SEQ ID NO:25) and/orAla-Ala/Gly-Leu-Leu-His-Asp-Thr-Val-Glu-Asp-Thr (SEQ ID NO:26) and/orThr-Pro-Val-Asp-Phe-Ala-Tyr-Ala-Val-His-Thr-Glu-Val-Gly-His-Arg (SEQ IDNO:27) are not disrupted by such insertions/deletions.

The invention relates to an isolated polynucleotide comprising thenucleotide sequence according to SEQ ID NO:5 or 7, adenine beingoptionally present in position 785 of SEQ ID NO:5 or position 1535 ofSEQ ID NO:7.

The invention relates to an isolated polynucleotide comprising the relallele of the DM1915 mutant.

The invention relates to an isolated polynucleotide comprising part ofthe coding region of a rel allele of the invention, said isolatedpolynucleotide comprising in any case that part of the coding regionwhich comprises the amino acid substitution in position 38 of the aminoacid sequence of the encoded polypeptide.

A nucleic acid molecule or DNA fragment comprises a molecule or fragmentwhich encodes at least one amino acid sequence corresponding topositions 19 to 57 of SEQ ID NO:2 or which encodes at least one aminoacid sequence corresponding to positions 9 to 107 of SEQ ID NO:2 orwhich encodes at least one amino acid sequence corresponding topositions 9 to 207 of SEQ ID NO:2 or which encodes at least one aminoacid sequence corresponding to positions 9 to 407 of SEQ ID NO:2 orwhich encodes at least one amino acid sequence corresponding topositions 9 to 607 of SEQ ID NO:2 or which encodes at least one aminoacid sequence corresponding to positions 9 to 707 of SEQ ID NO:2 orwhich encodes at least one amino acid sequence corresponding topositions 2 to 707 of SEQ ID NO:2, or which encodes at least one aminoacid sequence corresponding to positions 9 to 757 of SEQ ID NO:2 orwhich encodes at least one amino acid sequence corresponding topositions 2 to 757 of SEQ ID NO:2, or which encodes at least one aminoacid sequence corresponding to positions 2 to 758 of SEQ ID NO:2, orwhich encodes at least one amino acid sequence corresponding topositions 2 to 759 of SEQ ID NO:2, with one of the proteinogenic aminoacids other than L-proline, preferably L-leucine, being present in theposition corresponding to 38 of SEQ ID NO:2. L-Glutamic acid isoptionally present in position 262 of SEQ ID NO:2.

An example of a reading frame of the invention, comprising apolynucleotide encoding at least the amino acid sequence of positions 19to 57 corresponding to SEQ ID NO:2, with one of the proteinogenic aminoacids (Xaa) other than L-proline being present in the positioncorresponding to 38 of the amino acid sequence, is listed below:

gcc agg ctt gcc cgc agc ctc aca gga aac cgc gttAla Arg Leu Ala Arg Ser Leu Thr Gly Asn Arg Val    20                  25                  30cgc acc aac cct gtg ctg gat nnn ctg ctg agc atcArg Thr Asn Pro Val Leu Asp Xaa Leu Leu Ser Ile                35                  40cac cgg caa ttt cac cca cgc gcc gac gta caa gtgHis Arg Gln Phe His Pro Arg Ala Asp Val Gln Val        45                  50 ttg gaa cgt Leu Glu Arg 55

It is likewise depicted as SEQ ID NO:11. The amino acid sequence encodedby this reading frame is depicted as SEQ ID NO:12. Position 20 in SEQ IDNO:12 corresponds to position 38 of SEQ ID NO:2, 4, 6 or 8,respectively.

Nucleic acid molecules encoding at least one amino acid sequencecorresponding to positions 19 to 57 of SEQ ID NO:6 or 8, respectively,or at least corresponding to position 9 to 107 of SEQ ID NO:6 or 8,respectively, or at least corresponding to positions 9 to 207 of SEQ IDNO:6 or 8, respectively, or at least corresponding to positions 9 to 407of SEQ ID NO:6 or 8, respectively, or at least corresponding topositions 9 to 607 of SEQ ID NO:6 or 8, respectively, or at leastcorresponding to positions 9 to 707 of SEQ ID NO:6 or 8, respectively,or at least corresponding to positions 2 to 707 of SEQ ID NO: 6 or 8,respectively, or at least corresponding to positions 9 to 757 of SEQ IDNO: 6 or 8, respectively, or at least corresponding to positions 2 to757 of SEQ ID NO: 6 or 8, respectively, or at least corresponding topositions 2 to 758 of SEQ ID NO: 6 or 8, respectively, or at leastcorresponding to positions 2 to 759 of SEQ ID NO: 6 or 8, respectively,L-glutamic acid being optionally present in position 262 are preferred.

An example of a reading frame of the invention, comprising apolynucleotide encoding at least the amino acid sequence correspondingto positions 19 to 57 of SEQ ID NO:6 or 8, respectively, is listedbelow:

gcc agg ctt gcc cgc agc ctc aca gga aac cgc gttAla Arg Leu Ala Arg Ser Leu Thr Gly Asn Arg Val    20                  25                  30cgc acc aac cct gtg ctg gat ctg ctg ctg agc atcArg Thr Asn Pro Val Leu Asp Leu Leu Leu Ser Ile                35                  40cac cgg caa ttt cac cca cgc gcc gac gta caa gtgHis Arg Gln Phe His Pro Arg Ala Asp Val Gln Val        45                  50 ttg gaa cgt Leu Glu Arg 55

The reading frame is likewise depicted as SEQ ID NO:13. SEQ ID NO:14depicts the amino acid sequence encoded by said reading frame. Position20 in SEQ ID NO:14 corresponds to position 38 of SEQ ID NO:2, 4, 6 or 8,respectively.

Very particular preference is given to nucleic acid molecules comprisingat least one nucleotide sequence corresponding to positions 805 to 921of SEQ ID NO:7, or at least one nucleotide sequence corresponding topositions 655 to 1071 of SEQ ID NO:7, or at least one nucleotidesequence corresponding to positions 355 to 1371 of SEQ ID NO:7, or atleast one nucleotide sequence corresponding to positions 55 to 2671 ofSEQ ID NO:7, or at least one nucleotide sequence corresponding topositions 1 to 2725 of SEQ ID NO:7, adenine being optionally present inposition 1535.

In addition, the reading frames of the invention, as shown by way ofexample in SEQ ID NO:11 and 13 as nucleotide sequence and in SEQ IDNO:12 and SEQ ID NO:14 in the form of the encoded amino acid sequence,may comprise one or more mutations resulting in one or more conservativeamino acid substitutions. The mutations preferably result in no morethan 4%, no more than 2% or no more than 1%, conservative amino acidsubstitutions. The reading frames of the invention may furthermorecomprise one more silent mutations. The reading frames of the inventioncomprise preferably no more than 4%, and more preferably no more than 2%to no more than 1%, silent mutations.

The isolated polynucleotides of the invention may be used in order toproduce recombinant strains of microorganisms, which release amino acidsinto the surrounding medium or accumulate them in the cell interior inan improved manner, compared to the starting or parent strain.

A widespread method of incorporating mutations into genes of coryneformbacteria is that of allele substitution which is also referred to asgene replacement. This process involves transferring a DNA fragmentcomprising the mutation of interest into the desired strain of acoryneform bacterium and incorporating said mutation into the chromosomeof the desired strain by at least two recombination events or cross-overevents or replacing the sequence of a gene in the strain in questionwith the mutated sequence.

Schwarzer and Pühler (Bio/Technology 9, 84-87 (1991) used this method inorder to incorporate a lysA allele carrying a deletion and a lysA allelecarrying an insertion into the C. glutamicum chromosome, instead of thewild type gene. Schäfer et al. (Gene 145, 69-73 (1994)) employed saidmethod, in order to incorporate a deletion into the C. glutamicumhom-thrB operon. Nakagawa et al. (EP 1108790) and Ohnishi et al.(Applied Microbiology and Biotechnology 58(2), 217-223 (2002)) employedsaid method in order to incorporate various mutations, starting from theisolated alleles, into the C. glutamicum chromosome. In this way,Nakagawa et al. succeeded in incorporating a mutation referred to asVa159Ala into the homoserine dehydrogenase gene (hom), a mutationreferred to as Thr311Ile into the aspartate kinase gene (lysC and ask,respectively), a mutation referred to as Pro458Ser into the pyruvatecarboxylase gene (pyc) and a mutation referred to as Ala213Thr into theglucose 6-phoshate dehydrogenase gene (zwf) of C. glutamicum strains.

A process of the invention may use a polynucleotide of the invention,which comprises the entire coding region, as depicted, for example, inSEQ ID NO:5, or which comprises part of the coding region, such as, forexample, the nucleotide sequence encoding at least the amino acidsequence corresponding to positions 19 to 57 of SEQ ID NO:6 or 8,respectively, and depicted as SEQ ID NO:11 and 13. The part of thecoding region corresponding to SEQ ID NO:11 and 13 has a length of 117nucleobases. Preference is given to those parts of SEQ ID No:7 whichencompass at least the sequence between positions 1071 and 665, or atleast between positions 1371 and 355 and accordingly have a length of407 or 1017 nucleotides.

SEQ ID NO:15 shows an example of a polynucleotide of the invention whichencompasses a part of the coding region.

In said method, the DNA fragment comprising the mutation of interest istypically present in a vector, in particular a plasmid which preferablyis replicated only to a limited extent, if at all, by the strain to beprovided with the mutation. The auxiliary or intermediate host used, inwhich the vector can be replicated, may be a bacterium of the genusEscherichia, preferably of the species Escherichia coli.

Examples of plasmid vectors of this kind are the pK*mob and pK*mobsacBvectors described by Schäfer et al. (Gene 145, 69-73 (1994)), such as,for example, pK18mobsacB, and the vectors described in WO 02/070685 andWO 03/014362. These are replicative in Escherichia coli but not incoryneform bacteria. Suitable are vectors comprising a gene with aconditionally negative-dominant action, such as, for example, the sacBgene (levansucrase gene) of Bacillus, for example, or the galK gene(galactose kinase gene) of Escherichia coli, for example. (A gene withconditionally negative-dominant action means a gene which, under certainconditions, is disadvantageous, for example toxic, to the host but whichhas, under different conditions, no adverse effects on the host carryingthe gene.) Said vectors make possible the selection for recombinationevents in which the vector is eliminated from the chromosome. Nakamuraet al. (U.S. Pat. No. 6,303,383) furthermore described atemperature-sensitive plasmid for coryneform bacteria, which canreplicate only at temperatures below 31° C.

The vector is subsequently transferred to the coryneform bacterium byway of conjugation, for example by the method of Schäfer (Journal ofBacteriology 172, 1663-1666 (1990)), or transformation, for example bythe method of Dunican and Shivnan (Bio/Technology 7, 1067-1070 (1989))or the method of Thierbach et al. (Applied Microbiology andBiotechnology 29, 356-362 (1988)). The DNA may also be transferred,where appropriate, by particle bombardment.

Incorporation of the mutation is achieved after homologous recombinationby means of a first cross-over event causing integration and of asuitable second cross-over event causing excision in the target gene orin the target sequence, resulting in a recombinant bacterium.

The strains obtained may be identified and characterized by using, interalia, the methods of Southern blotting hybridization, polymerase chainreaction, sequence determination, the method of fluorescence resonanceenergy transfer (FRET) (Lay et al. Clinical Chemistry 43, 2262-2267(1997)) or methods of enzymology.

Accordingly, the invention further relates to a process for preparing acoryneform bacterium, which comprises

-   -   a) transferring a polynucleotide of the invention to a        coryneform bacterium,    -   b) replacing the GTP-pyrophosphate kinase gene which encodes an        amino acid sequence with L-proline in position 38 or in a        comparable position of said amino acid sequence and which is        present in the chromosome of said coryneform bacterium with the        polynucleotide of a), which encodes an amino acid sequence        having a different proteinogenic L-amino acid, preferably        L-leucine, in position 38 or in a comparable position of said        amino acid sequence, and    -   c) propagating the coryneform bacterium obtained by steps a) and        b).

In this way a recombinant coryneform bacterium is obtained whichcomprises one (1) rel allele of the invention, instead of the wild typerel gene.

Another process of the invention for preparing a microorganism comprises

-   -   a) transferring a polynucleotide of the invention, which encodes        a polypeptide having GTP-pyrophosphate kinase enzyme activity,        to a microrganism,    -   b) replicating said polynucleotide in said microorganism, and    -   c) propagating the microorganism obtained by steps a) and b).

In this way, a recombinant microorganism is obtained, which comprises atleast one (1) copy or several copies of a polynucleotide of theinvention, which polynucleotide encodes a GTP-pyrophosphate kinasecomprising one of the proteinogenic amino acids other than L-proline inposition 38 or a comparable position of the amino acid sequence of theencoded polypeptide, the substitution with L-leucine being preferred.

The invention further relates to hosts or host cells, preferablymicroorganisms, more preferably coryneform bacteria and bacteria of thegenus Escherichia, which comprise the polynucleotides of the invention.The invention likewise relates to microorganisms prepared by using theisolated polynucleotides. Such microorganisms or bacteria are alsoreferred to as recombinant microorganisms or recombinant bacteria. Inthe same way, the invention relates to vectors comprising thepolynucleotides of the invention. Finally, the invention likewiserelates to hosts harboring said vectors.

The isolated polynucleotides of the invention may likewise be used forachieving overexpression of the polypeptides encoded by them.

Overexpression means an increase in the intracellular concentration oractivity of a ribonucleic acid, a protein or an enzyme. In thisinvention, rel alleles or polynucleotides which encode GTP-pyrophosphatekinases comprising one of the proteinogenic amino acids other thanL-proline in position 38 of the amino acid sequence of the encodedpolypeptide, with the substitution with L-leucine being preferred, areoverexpressed. Enzymes endogenous to the host—“aminopeptidases”—areknown to be able to cleave N-terminal amino acids, in particular theN-terminal methionine, off the polypeptide produced. Said increase inthe concentration or activity of a gene product can be achieved, forexample, by increasing the copy number of the correspondingpolynucleotides by at least one copy.

A method of increasing the copy number comprises incorporating theappropriate gene or allele into a vector, preferably a plasmid, which isreplicated by a coryneform bacterium. Examples of suitable plasmidvectors are pZ1 (Menkel et al., Applied and Environmental Microbiology(1989) 64: 549-554) or the pSELF vectors described by Tauch et al.(Journal of Biotechnology 99, 79-91 (2002)). A review article onplasmids in Corynebacterium glutamicum can be found in Tauch et al.(Journal of Biotechnology 104, 27-40 (2003)).

Another common method of achieving overexpression is the process ofchromosomal gene amplification. This method involves inserting at leastone additional copy of the gene or allele of interest into thechromosome of a coryneform bacterium.

For the hom-thrB operon in Reinscheid et al. (Applied and EnvironmentalMicrobiology 60, 126-132 (1994)), a plasmid which is non-replicative inC. glutamicum and which comprises the gene of interest is transferred toa coryneform bacterium. After homologous recombination by means of across-over event, the resulting strain comprises at least two copies ofthe gene or allele in question.

WO 03/040373 and US-2003-0219881-A1 describe that one or more copies ofthe gene of interest are inserted at a desired side of the C. glutamicumchromosome by means of at least two recombination events. In this way,for example, a copy of a lysC allele encoding a L-lysine-insensitiveaspartate kinase was incorporated into the C. glutamicum gluB gene.

WO 03/014330 and US-2004-0043458-A1 describe that at least one furthercopy, preferably in tandem arrangement to the gene or allele alreadypresent, of the gene of interest is incorporated by means of at leasttwo recombinantion events at the natural locus. In this way it waspossible, for example, to achieve a tandem duplication of a lysC^(FBR)allele at the natural lysC gene locus.

Another method of achieving overexpression comprises linking theappropriate gene or allele functionally (operably linked) to a promoteror an expression cassette. Examples of suitable promotors forCorynebacterium glutamicum are described in the review article by Pateket al. (Journal of Biotechnology 104(1-3), 311-323 (2003)). In a similarmanner, the variants of the dapA promotor described by Vasicova et al(Journal of Bacteriology 181, 6188-6191 (1999)) may be used, for examplethe promotor A25. The gap promotor of Corynebacterium glutamicum (EP06007373) may also be used. It is finally possible to use the well-knownpromotors T3, T7, SP6, M13, lac, tac and trc described by Amann et al.(Gene 69(2), 301-315 (1988)) and Amann and Brosius (Gene 40(2-3),183-190 (1985)). Such a promotor may be inserted, for example, upstreamof the rel allele, at a distance of approximately 1-500 nucleotides fromthe start codon, of a recombinant coryneform bacterium, which alleleencodes a protein, which comprises, instead of the amino acid L-prolinenaturally present in position 38, a different proteinogenic amino acid.A promotor of this kind may naturally likewise be inserted upstream ofthe coding region of the rel allele of a mutant of the invention. It isfurthermore possible to link an isolated polynucleotide of theinvention, which encodes a variant of the invention of GTP-pyrophosphatekinase, to a promotor and to incorporate the expression unit obtainedinto an extrachromosomally replicating plasmid or into the chromosome ofa coryneform bacterium.

In addition, it is possible to mutate the promotor and regulatoryregions or the ribosomal binding site which is located upstream of thestructural gene. Measures of extending the mRNA lifetime likewiseimprove expression. Preventing the degradation of the enzyme proteinfurthermore likewise enhances enzyme activity. Alternatively, the geneor allele in question may furthermore be overexpressed by altering themedia composition and the culturing process.

The overexpression measures increase the activity or concentration ofthe protein in question by at least 10%, 25%, 50%, 75%, 100%, 150%,200%, 300%, 400% or 500%, up to no more than 1000% or 2000%, based onthe activity or concentration of the protein in the startingmicroorganism or parent strain. A starting microorganism or parentstrain means a microorganism which is subjected to the measures of theinvention.

The concentration of the protein may be determined via 1- and2-dimensional protein gel fractionation and subsequent opticalidentification of the protein concentration in the gel, usingappropriate evaluation software. A common method of preparing theprotein gels in the case of coryneform bacteria and of identifying theproteins is the procedure described by Hermann et al. (Electrophoresis,22:1712-23 (2001)). The protein concentration may likewise be determinedby Western blot hybridization using an antibody specific for the proteinto be detected (Sambrook et al., Molecular cloning: a laboratory manual.2^(nd) Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989) and subsequent optical evaluation using appropriateconcentration determination software (Lohaus and Meyer (1998)Biospektrum 5:32-39; Lottspeich, Angewandte Chemie 38: 2630-2647(1999)).

The invention relates to processes for overexpressing theGTP-pyrophosphate kinases of the invention. A process of the inventionfor overexpression comprises, inter alia, increasing the copy number ofa polynucleotide of the invention, which polynucleotide encodes aGTP-pyrophosphate-kinase variant comprising one of the proteinogenicamino acids other than L-proline in position 38 or the correspondingposition of the encoded amino acid sequence, by at least one (1) orseveral copies. Another process of the invention comprises functionallylinking a promotor to the polynucleotide.

The invention furthermore relates to microorganisms having an increasedconcentration or activity of the GTP-pyrophosphate kinase variants ofthe invention in their cell interior.

For improved production of L-amino acids different genes in the mutantsor recombinant strains of the invention may be overexpressed, preferablyendogenous genes.

“Endogenous genes” or “endogenous nucleotide sequences” means the genesor the nucleotide sequences or alleles present in the population of aspecies.

Thus it is possible to overexpress for the preparation of L-lysine oneor more of the genes selected from the group consisting of

-   -   a dapA gene encoding a dihydrodipicolinate synthase (DapA, EC        No. 4.2.1.52), such as, for example, the Corynebacterium        glutamicum wild-type dapA gene described in EP 0 197 335,    -   a lysA gene encoding a diaminopimelate decarboxylase (LysA, EC        No. 4.1.1.20), such as, for example, the Corynebacterium        glutamicum lysA gene ATCC13869, described in U.S. Pat. No.        6,090,597,    -   a zwf gene encoding a glucose-6-phosphate dehydrogenase (Zwf, EC        No. 1.1.1.49) such as, for example, the Corynebacterium        glutamicum wild-type zwf gene described in JP-A-09224661 and        EP-A-1108790,    -   the Corynebacterium glutamicum zwf alleles described in        US-2003-0175911-A1, which encode a protein having glucose        6-phosphate dehydrogenase activity in which, for example, the        L-alanine in position 243 of the amino acid sequence has been        replaced with L-threonine or in which the L-aspartic acid in        position 245 has been replaced with L-serine,    -   a pyc gene encoding a pyruvate carboxylase (Pyc, EC No.        6.4.1.1), such as, for example, the Corynebacterium glutamicum        wild-type pyc gene described in DE-A-198 31 609 and EP 1108790,    -   the Corynebacterium glutamicum pyc allele described in EP 1 108        790, which encodes a protein having pyruvate carboxylase        activity in which L-proline in position 458 of the amino acid        sequence has been replaced by L-serine,    -   the Corynebacterium glutamicum pyc alleles described in WO        02/31158 and in particular EP1325135B1, which encode proteins        having pyruvate carboxylate activity which carry one or more of        the amino acid substitutions selected from the group consisting        of L-valine in position 1 replaced with L-methionine, L-glutamic        acid in position 153 replaced with L-aspartic acid, L-alanine in        position 182 replaced with L-serine, L-alanine in position 206        replaced with L-serine, L-histidine in position 227 replaced        with L-arginine, L-alanine in position 455 replaced with glycine        and L-aspartic acid in position 1120 replaced with L-glutamic        acid,    -   a lysC gene encoding an aspartate kinase (LysC, EC No. 2.7.2.4),        such as, for example, that of Corynebacterium glutamicum        wild-type lysC gene described as SEQ ID NO:281 in EP-A-1108790        (see also accession numbers AX120085 and 120365) and that of        Corynebacterium glutamicum wild-type lysC gene, described as SEQ        ID NO:25 in WO 01/00843 (see accession number AX063743),    -   a lysC^(FBR) allele, in particular according to Table 1, which        encodes a feedback-resistant aspartate kinase variant,    -   a lysE gene encoding a lysine export protein (LysE), such as,        for example, the Corynebacterium glutamicum wild-type lysE gene        described in DE-A-195 48 222,    -   the aat gene encoding an aspartate amino transferase (Aat, EC        No. 2.6.1.1) (the aat gene of Corynebacterium glutamicum        ATCC13032 is described, for example, in Kalinowski et al        (Journal of Biotechnology 104 (1-3), 5-25 (2003); see also        Accession number NC_(—)006958). It is referred to there as aspB        gene. U.S. Pat. No. 6,004,773 refers to a gene encoding an        aspartate amino transferase as aspC. Marienhagen et al (Journal        of Bacteriology 187 (22), 7693-7646 (2005) refer to the aat gene        as aspT gene.)),    -   the Corynebacterium glutamicum wild-type zwa1 gene encoding the        Zwa1 protein (U.S. Pat. No. 6,632,644).

In addition to using the alleles of the invention of the rel gene, forthe purpose of producing L-lysine, to simultaneously attenuation oreliminate, where appropriate, may be obtained with simultaneousoverexpression of at least one of the genes selected from theabovementioned group of genes, one or more of the endogenous genesselected from the group consisting of

-   -   a pgi gene encoding glucose 6-phosphate isomerase (Pgi, EC No.        5.3.1.9), such as, for example, the Corynebacterium glutamicum        pgi gene described in U.S. Pat. No. 6,586,214 and U.S. Pat. No.        6,465,238,    -   a hom gene encoding homoserine dehydrogenase (Horn, EC No.        1.1.1.3), such as, for example, the Corynebacterium glutamicum        hom gene described in EP-A-0131171,    -   a thrB gene encoding homoserine kinase (ThrB, EC No. 2.7.1.3.9),        such as, for example, the Corynebacterium glutamicum thrB gene        described by Peoples et al. (Molecular Microbiology 2 (1988):        63-72)), and    -   a pfkB gene encoding phosphofructokinase (PfkB, EC No.        2.7.1.56), such as, for example, the Corynebacterium glutamicum        pfkB gene described in WO 01/00844 (sequence no. 57),    -   an mdh gene encoding malate dehydrogenase (Mdh, EC No.        1.1.1.37), as described, for example, in WO 02/02778,    -   an mqo gene coding for malate-quinone oxidoreductase (Mqo, EC        No. 1.1.99.16), as described, for example, in U.S. Pat. No.        7,094,106 and PCT/EP2005/057216.

In this connection, the term “attenuation” describes the reduction orelimination of the intracellular activity of one or more enzymes(proteins) which are encoded by the corresponding DNA in a microorganismwhich is achieved, for example, by using a weak promoter or using a geneor allele which encodes a corresponding enzyme having low activity, orinactivating the corresponding gene or enzyme (protein), and, whereappropriate, combining these measures.

As a result of using the measures for achieving attenuation, theactivity or concentration of the corresponding protein is lowered tofrom 0 to 75%, from 0 to 50%, from 0 to 25%, from 0 to 10%, or from 0 to5%, of the activity or concentration of the wild-type protein or of theactivity or concentration of the protein in the starting microorganism.

Mutations which come into consideration for generating an attenuationare transitions, transversions, insertions and deletions of at least one(1) base pair or nucleotide. Depending on the effect which the aminoacid substitution elicited by the mutation has on the enzyme activity,reference is made to missense mutations or nonsense mutations. Amissense mutation leads to the replacement of a given amino acid in aprotein with another amino acid, with the amino acid replacementconstituting, in particular, a nonconservative amino acid substitution.This substitution impairs the efficiency or activity of the protein andreduces it down to a value of from 0 to 75%, from 0 to 50%, from 0 to25%, from 0 to 10%, or from 0 to 5%. A nonsense mutation leads to a stopcodon being located in the coding region of the gene and consequently totranslation being terminated prematurely. Insertions or deletions of atleast one base pair in a gene lead to frame shift mutations which resultin incorrect amino acids being incorporated or in the translation beingterminated prematurely. If a stop codon is formed in the coding regionas a consequence of mutation, this then also leads to translation beingterminated prematurely. Said measures are preferably carried out in the5′-terminal part of the coding region, which encodes the N terminus ofthe polypeptide. If the overall length of a polypeptide (measured as thenumber of chemically linked L-amino acids) is set to 100%, then thatpart of the amino acid sequence, which, counted from the start aminoacid L-formyl methionine, comprises 80% of the subsequent L-amino acids,belongs to the N terminus of the polypeptide.

Further directions for generating such mutations belong to the prior artand are contained in known textbooks of genetics and molecular biologysuch as the textbook by Knippers (“Molekulare Genetik [MolecularGenetics]”, 6^(th) edition, Georg Thieme Verlag, Stuttgart, Germany,1995), that by Winnacker (“Gene und Klone [Genes and Clones]”, VCHVerlagsgesellschaft, Weinheim, Germany, 1990) or that by Hagemann(“Allgemeine Genetik [General Genetics]”, Gustav Fischer Verlag,Stuttgart, 1986). Further measures are described in the prior art.

One method of reducing gene expression involves putting the gene to beattenuated under the control of a promotor inducible by addition ofmetered amounts of IPTG (isopropyl-β-D-thiogalactopyranoside), such as,for example, the trc promotor or the tac promotor. Examples of suitablevectors here are the Escherichia coli expression vector pXK99E(WO0226787; deposited in accordance with the Budapest Treaty inDH5alpha/pXK99E as DSM14440 with the Deutschen Sammlung fürMikroorganismen und Zellkulturen [German collection of microorganismsand cell cultures] (DSMZ, Braunschweig, Germany) on 31 Jul. 2001) andpVWEx2 (Wendisch, pH. D. thesis, Berichte des Forschungszentrums Jülich,Jül-3397, ISSN 0994-2952, Jëlich, Germany (1997)), which vectors enablethe cloned gene to be expressed in Corynebacterium glutamicum in anIPTG-dependent manner.

This method was employed, for example, in the patent WO0226787 forregulated expression of the deaD gene by integrating the pXK99EdeaDvector into the Corynebacterium glutamicum genome, and by Simic et al.(Applied and Environmental Microbiology 68: 3321-3327 (2002)) forregulated expression of the glyA gene by integrating the pK18mobglyA′vector in Corynebacterium glutamicum.

Another method of specifically reducing gene expression is the antisensetechnique which involves delivering oligodeoxynucleotides or vectors tothe target cells in order to synthesize relatively long antisense RNA.Said antisense RNA may bind there to complementary sections of specificmRNAs and reduce their stability or block translatability. An example ofthis can be found by the skilled worker in Srivastava et al. (AppliedEnvironmental Microbiology 2000 October; 66 (10): 4366-4371).

The isolated coryneform bacteria which are obtained by the measures ofthe invention exhibit a secretion or production of the desired aminoacid, in a fermentation process, which is increased as compared withthat of the starting strain or parent strain which was initiallyemployed.

“Isolated bacteria” are to be understood as being the mutants andrecombinant bacteria, in particular coryneform bacteria, according tothe invention which are isolated or generated and which comprise a relallele which encodes a GTP-pyrophosphate kinase which comprises thedescribed amino acid substitution in position 38 of the amino acidsequence.

The performance of the isolated bacteria, or of the fermentation processwhen using these bacteria, in regard to one or more of the parametersselected from the group comprising the product concentration (productper volume), the product yield (product formed per carbon sourceconsumed) and the product formation (product formed per volume andtime), or else of other process parameters and combinations, is improvedby at least 0.5%, at least 1%, at least 1.5%, or at least 2%, based onthe starting strain or parent strain or the fermentation process whenusing these strains.

The isolated coryneform bacteria according to the invention can becultured continuously, as described, for example, in PCT/EP2004/008882,or discontinuously, in a batch process or a fed-batch process or arepeated fed-batch process, for the purpose of producing L-amino acids.A general summary of known culturing methods can be found in thetextbook by Chmiel (Bioprozesstechnik 1. Einführung in dieBioverfahrenstechnik [Bioprocess Technology 1. Introduction toBioprocess Technology] (Gustav Fischer Verlag, Stuttgart, 1991)) or inthe textbook by Storhas (Bioreaktoren and periphere Einrichtungen[Bioreactors and Peripheral Equipment] (Vieweg Verlag,Brunswick/Wiesbaden, 1994)).

The culture medium or fermentation medium to be used must suitablysatisfy the requirements of the given strains. Descriptions of media forculturing different microorganisms are given in the manual “Manual ofMethods for General Bacteriology” published by the American Society forBacteriology (Washington D.C., USA, 1981). The terms culture medium,fermentation medium and feed medium or medium are mutuallyinterchangeable.

The carbon source employed can be sugars and carbohydrates, such asglucose, sucrose, lactose, fructose, maltose, molasses,sucrose-containing solutions derived from sugar beet or sugar caneproduction, starch, starch hydrolysate and cellulose, oils and fats,such as soybean oil, sunflower oil, peanut oil and coconut oil, fattyacids, such as palmitic acid, stearic acid and linoleic acid, alcohols,such as glycerol, methanol and ethanol, and organic acids, such asacetic acid. These substances can be used individually or as mixtures.

The nitrogen source employed can be organic nitrogen-containingcompounds, such as peptones, yeast extract, meat extract, malt extract,cornsteep liquor, soybean flour and urea, or inorganic compounds, suchas ammonium sulfate, ammonium chloride, ammonium phosphate, ammoniumcarbonate and ammonium nitrate. The nitrogen sources can be usedindividually or as mixtures.

The phosphorus source employed can be phosphoric acid, potassiumdihydrogen phosphate or dipotassium hydrogen phosphate or thecorresponding sodium-containing salts.

The culture medium must furthermore contain salts, for example in theform of chlorides or sulfates of metals such as sodium, potassium,magnesium, calcium and iron, for example magnesium sulfate or ironsulfate, which are necessary for growth. Finally, growth substances,such as amino acids, for example homoserine, and vitamins, for examplethiamine, biotin or pantothenic acid, can be used in addition to theabovementioned substances. In addition to this, suitable precursors ofthe respective amino acid can be added to the culture medium.

The abovementioned added substances can be added to the culture in theform of a once-only mixture or fed in a suitable manner during theculture.

Basic compounds such as sodium hydroxide, potassium hydroxide, ammoniaor ammonia water, or acidic compounds such as phosphoric acid orsulfuric acid, are employed in a suitable manner for controlling the pHof the culture. In general, the pH is adjusted to a value of from 6.0 to9.0, preferably of from 6.5 to 8. It is possible to use antifoamants,such as fatty acid polyglycol esters, for controlling foam formation.Suitable substances which act selectively, such as antibiotics, can beadded to the medium in order to maintain the stability of plasmids. Inorder to maintain aerobic conditions, oxygen or oxygen-containing gasmixtures, such as air, are passed into the culture. It is also possibleto use liquids which are enriched with hydrogen peroxide. Whereappropriate, the fermentation is conducted under positive pressure, forexample under a pressure of 0.03 to 0.2 MPa. The temperature of theculture is normally from 20° C. to 45° C., and preferably from 25° C. to40° C. In the case of batch processes, the culture is continued until amaximum of the desired amino acid has been formed. This objective may beachieved within from 10 hours to 160 hours. Longer culturing times arepossible in the case of continuous processes.

Suitable fermentation media are described, inter alia, in U.S. Pat. No.6,221,636, U.S. Pat. No. 5,840,551, U.S. Pat. No. 5,770,409, U.S. Pat.No. 5,605,818, U.S. Pat. No. 5,275,940, U.S. Pat. No. 4,275,157 and U.S.Pat. No. 4,224,409.

Methods for determining L-amino acids are disclosed in the prior art.The analysis can, for example, take place by means of anion exchangechromatography, followed by ninhydrin derivatization, as described inSpackman et al. (Analytical Chemistry, 30 (1958), 1190), or it can takeplace by reversed phase HPLC, as described in Lindroth et al.(Analytical Chemistry (1979) 51: 1167-1174).

The invention accordingly relates to a process for preparing an L-aminoacid, which comprises

-   a) fermenting an isolated coryneform bacterium in a suitable medium,    said bacterium comprising a gene encoding a polypeptide having    GTP-pyrophosphate kinase enzyme activity, with the L-proline in    position 38 or the corresponding position in the amino acid    sequences of said polypeptide having been replaced by a different    proteinogenic amino acid, preferably L-leucine, and-   b) the L-amino acid being accumulated in the fermentation broth or    in the cells of the isolated coryneform bacterium.

For this purpose, the L-amino acid accumulated in the nutrient medium orin the fermentation broth and/or in the bacterial cells are usuallycollected in order to obtain a solid or liquid product.

A fermentation broth is understood as being a fermentation medium inwhich a microorganism is cultured for a certain time and at a certaintemperature. The fermentation medium, and/or the medium employed duringthe fermentation, contains/contain all the substances or componentswhich ensure propagation of the microorganism and the formation of thedesired amino acid.

At the conclusion of the fermentation, the resulting fermentation brothaccordingly contains a) the biomass (cell mass) of the microorganismwhich has been formed as a consequence of the propagation of the cellsof the microorganism, b) the desired amino acid which has been formedduring the fermentation, c) the organic by-products which have beenformed during the fermentation, and d) the constituents of thefermentation medium/fermentation media employed, or the addedsubstances, for example vitamins, such as biotin, amino acids, such ashomoserine, or salts, such as magnesium sulfate, which were not consumedby the fermentation.

The organic by-products include substances which are produced by themicroorganisms employed in the fermentation, where appropriate inaddition to the given desired L-amino acid, and are secreted, whereappropriate. These by-products include L-amino acids which amount toless than 30%, 20% or 10% compared with the desired amino acid. Theyalso include organic acids which carry from one to three carboxylgroups, such as acetic acid, lactic acid, citric acid, malic acid orfumaric acid. They also include sugars, such as trehalose.

Fermentation broths which are suitable for industrial purposes may havean amino acid content of from 30 g/kg to 200 g kg or of from 40 g/kg to175 g/kg or of from 50 g/kg to 150 g/kg. The content of biomass (as drybiomass) may be from 20 to 50 g/kg.

In the case of the amino acid L-lysine, four different product formshave been disclosed in the prior art.

One group of L-lysine-containing products comprises concentrated,aqueous, alkaline solutions of purified L-lysine (EP-B-0534865). Anothergroup, as described, for example, in U.S. Pat. No. 6,340,486 and U.S.Pat. No. 6,465,025, comprises aqueous, acidic, biomass-containingconcentrates of L-lysine-containing fermentation broths. The best-knowngroup of solid products comprises pulverulent or crystalline forms ofpurified or pure L-lysine, which is present in the form of a salt suchas L-lysine monohydrochloride. Another group of solid product forms isdescribed, for example, in EP-B-0533039. The product form which isdescribed in this document contains, in addition to L-lysine, the majorportion of the added substances which were used during the fermentativepreparation, and which were not consumed, and, where appropriate,from >0% to 100% of the biomass of the microorganism employed.

In the case of the amino acids L-valine, L-isoleucine, L-proline,L-tryptophan and L-homoserine, product form containing the amino acidsin question in purified or pure form (=95% by weight of =98% by weight)are known in the art.

In correspondence with the different product forms, a very wide varietyof methods are known for collecting, isolating or purifying the L-aminoacid from the fermentation broth for the purpose of preparing theL-amino acid-containing product or the purified L-amino acid.

Ion exchange chromatography methods, using active charcoal, andcrystallization methods are used for preparing solid, pure L-aminoacids. In the case of lysine, using these methods results in thecorresponding base or a corresponding salt such as the monohydrochloride(Lys-HCl) or the lysine sulfate (Lys₂-H₂SO₄).

As far as lysine is concerned, EP-B-0534865 describes a method forpreparing aqueous, basic L-lysine-containing solutions from fermentationbroths. In this document, the biomass is separated off from thefermentation broth and discarded. A base such as sodium hydroxide,potassium hydroxide or ammonium hydroxide is used to adjust the pH tobetween 9 and 11. Following concentration and cooling, the mineralconstituents (inorganic salts) are separated off from the broth bycrystallization and either used as fertilizer or discarded.

In the case of processes for preparing lysine using the bacteriaaccording to the invention, use is also made of those processes whichresult in products which contain constituents of the fermentation broth.These products are, in particular, used as animal feed additives.

Depending on the requirement, the biomass can be entirely or partiallyremoved from the fermentation broth by means of separation methods suchas centrifugation, filtration or decanting, or a combination of thesemethods, or all the biomass can be left in the fermentation broth. Whereappropriate, the biomass, or the biomass-containing fermentation broth,is inactivated during a suitable process step, for example by means ofthermal treatment (heating) or by means of adding acid.

The chemical components of the biomass are inter alia the cell envelope,for example peptidoglycan and arabinogalactane, the protein orpolypeptides, for example the GTP-pyrophosphate kinase polypeptide,lipids and phospholipids and nucleic acids (DNA and RNA), for examplepolynucleotides comprising the mutation of the invention. As aconsequence of the inactivation measures and/or the other steps (forexample acidification, spray drying, granulation etc.), nucleic acidstypically are fragments having a length of, inter alia, =40-60bp, >60-80 bp, >80-100 bp, >100-200 bp, >200-300 bp, >300-400bp, >400-500 bp, >500-750 bp, >750-1000 bp, >1000-1250 bp, >1250-1500bp, >1500-1750 bp, >1750-2000 bp, >2000-2500 bp, >2500-3000bp, >3000-4000 bp, >4000-5000 bp.

In one embodiment, the biomass is completely or virtually completelyremoved, such that no (0%) or at most 30%, at most 20%, at most 10%, atmost 5%, at most 1% or at most 0.1%, of the biomass remains in theprepared product. In another embodiment, the biomass is not removed, oronly removed in trivial amounts, such that all (100%) or more than 70%,80%, 90%, 95%, 99% or 99.9% of the biomass remains in the preparedproduct. In one process according to the invention, the biomass isaccordingly removed in proportions of from =0% to =100%.

The fermentation broth which is obtained after the fermentation can beadjusted, before or after the biomass has been completely or partiallyremoved, to an acid pH using an inorganic acid, such as hydrochloricacid, sulfuric acid or phosphoric acid, or an organic acid, such aspropionic acid (GB 1,439,728 or EP 1 331 220). It is likewise possibleto acidify the fermentation broth when it contains the entire biomass(U.S. Pat. No. 6,340,486 or U.S. Pat. No. 6,465,025). The broth can alsobe stabilized by adding sodium bisulfite (NaHSO₃, GB 1,439,728) oranother salt, for example an ammonium, alkali metal or alkaline earthmetal salt of sulfurous acid.

Organic or inorganic solids which may be present in the fermentationbroth are partially or entirely removed when the biomass is separatedoff. At least some (>0%), preferably at least 25%, preferably at least50%, and more preferably at least 75%, of the organic by-products whichare dissolved in the fermentation broth and the constituents of thefermentation medium (added substances), which are dissolved and notconsumed remain in the product. Where appropriate, these by-products andconstituents also remain completely (100%) or virtually completely, thatis >95% or >98%, in the product. In this sense, the term “fermentationbroth basis” means that a product comprises at least a part of theconstituents of the fermentation broth.

Subsequently, water is extracted from the broth, or the broth isthickened or concentrated, using known methods, for example using arotary evaporator, a thin-film evaporator or a falling-film evaporator,or by means of reverse osmosis or nanofiltration. This concentratedfermentation broth can then be worked up into flowable products, inparticular into a finely divided powder or, preferably, a coarse-grainedgranulate, using methods of freeze drying, of spray drying or of spraygranulation, or using other methods, for example in a circulatingfluidized bed as described in PCT/EP2004/006655. Where appropriate, adesired product is isolated from the resulting granulate by means ofscreening or dust separation.

It is possible to dry the fermentation broth directly, i.e. by spraydrying or spray granulation without any prior concentration.

“Flowable” is understood as meaning powders which discharge unhinderedfrom a series of glass discharge vessels having discharge apertures ofdifferent sizes, i.e. which discharge unhindered at least from thevessel having a 5 mm (millimeter) aperture (Klein: Seifen, Öle, Fette,Wachse [Soaps, Oils, Fats and Waxes] 94, 12 (1968)).

“Finely divided” means a powder the majority (>50%) of which has aparticle size which is from 20 to 200 μm in diameter.

“Coarse-grained” means a product the majority (>50%) of which has aparticle size of from 200 to 2000 μm in diameter.

The particle size can be determined using methods of laser diffractionspectrometry. The corresponding methods are described in the textbook“Teilchengröβenmessung in der Laborpraxis [Particle Size Measurement inLaboratory Practice]” by R. H. Müller and R. Schuhmann,Wissenschaftliche Verlagsgesellschaft Stuttgart (1996) or in thetextbook “Introduction to Particle Technology” by M. Rhodes, Wiley &Sons (1998).

The flowable, finely divided powder can in turn be converted, by meansof suitable compacting or granulating methods, into a coarse-grained,readily flowable, storable, and to a large extent dust-free, product.

The term “dust-free” means that the product only contains smallproportions (<5%) of particle sizes of less than 100 μm in diameter.

Within the meaning of this invention, “storable” means a product whichcan be stored for at least one (1) year or longer, preferably at least1.5 years or longer, particularly preferably two (2) years or longer, ina dry and cool environment without there being any significant loss(<5%) of the given amino acid.

The invention also relates to a process for preparing an L-amino acid-,preferably L-lysine- or L-tryptophan-, containing product, preferably ananimal feed additive, from fermentation broths, which process ischaracterized by the steps of

-   a) culturing and fermenting an L-amino acid-secreting coryneform    bacterium, which comprises at least one rel allele encoding a    polypeptide having GTP-pyrophosphate kinase activity, which    polypeptide comprises an amino acid sequence in which one of the    proteinogenic amino acids other than L-proline, preferably    L-leucine, is present in position 38 or the comparable position, in    a fermentation medium,-   b) removing from 0 to 100% by weight of the biomass which is formed    during the fermentation, and-   c) drying the fermentation broth which is obtained in accordance    with a) and/or b) in order to obtain the product in the desired    powder form or granulate form,

with, where appropriate, an acid selected from the group sulfuric acid,phosphoric acid or hydrochloric acid being added prior to step b) or c).Preferably, water is being removed (concentration) from the L-aminoacid-containing fermentation broth after step a) or b).

The invention further relates to a process for preparing a lysinesulfate-containing product, which process is outlined in DE 102006016158and which involves processing the fermentation broth obtained using themicroorganisms of the invention, from which broth the biomass hasoptionally been removed, either completely or partially, by carrying outa process comprising at least the following steps:

-   a) lowering the pH by adding sulfuric acid to from 4.0 to 5.2, in    particular 4.9 to 5.1, and establishing in the broth a molar    sulfate/L-lysine ratio from 0.85 to 1.2, preferably 0.9 to 1.0, more    preferably >0.9 to <0.95, where appropriate by adding another or a    plurality of sulfate-containing compounds, and-   b) concentrating and, where appropriate, granulating the mixture    obtained in this way by removing water,    -   where appropriate carrying out, prior to step a), either or both        of the following measures:-   c) measuring the molar ratio of sulfate/L-lysine in order to    determine the required amount of sulfate-containing compounds-   d) adding a sulfate-containing compound selected from the group    consisting of ammonium sulfate, ammonium hydrogen sulfate and    sulfuric acid in appropriate ratios.

Where appropriate, a salt of sulfurous acid, preferably alkali metalhydrogen sulfite, more preferably sodium hydrogen sulfite, is added in aconcentration of from 0.01 to 0.5% by weight, preferably 0.1 to 0.3% byweight, more preferably 0.1 to 0.2% by weight, based on the fermentationbroth, prior to step b).

Preferred sulfate-containing compounds are ammonium sulfate and/orammonium hydrogen sulfate or corresponding mixtures of ammoniac andsulfuric acid and sulfuric acid itself.

The molar sulfate/L-lysine ratio V is calculated according to theformula: V=2×[SO₄ ²⁻]/[L-lysine]. This formula takes into account thefact that the SO₄ ²⁻ anion is divalent. A ratio of V=1 means thatLys₂(SO₄) has a stoichiometric composition, while a 10% sulfateshortfall is found at a ratio of V=0.9 and a 10% sulfate excess is foundat a ratio of V=1.1.

Customary organic or inorganic auxiliary substances, or carriersubstances such as starch, gelatin, cellulose derivatives or similarsubstances, may be used as binders, gelatinizers or thickeners infoodstuff or feedstuff processing, or other substances, such as silicicacids, silicates (EP0743016A) or stearates, in connection with thegranulation or compacting.

The surface of the resulting granulates are provided with oils, asdescribed in WO 04/054381. The oils which can be used are mineral oils,vegetable oils or mixtures of vegetable oils. Examples of these oils aresoybean oil, olive oil and soybean oil/lecithin mixtures. In the sameway, silicone oils, polyethylene glycols or hydroxyethyl cellulose alsocan be used. Treating the surfaces with said oils increases the abrasionresistance of the product and reduces the dust content. The content ofoil in the product is from 0.02 to 2.0% by weight, preferably from 0.02to 1.0% by weight, and more preferably from 0.2 to 1.0% by weight, basedon the total quantity of the feedstuff additives.

Products having a content of =97% by weight of a particle size of from100 to 1800 μm, or a content of =95% by weight of a particle size offrom 300 to 1800 μm, in diameter are preferred. The content of dust,i.e. particles having a particle size of <100 μm, is preferably from >0to 1% by weight, more preferably at most 0.5% by weight.

Alternatively, the product can also be absorbed onto an organic orinorganic carrier substance which is known in feedstuff processing, forexample silicic acids, silicates, grists, brans, meals, starches, sugarsetc., and/or be mixed and stabilized with thickeners or binders.Application examples and methods in this regard are described in theliterature (Die Mühle+Mischfuttertechnik [The Grinding Mill+Mixed FeedTechnology] 132 (1995) 49, page 817).

The product can also be brought, by means of coating methods using filmformers such as metal carbonates, silicic acids, silicates, alginates,stearates, starches, rubbers and cellulose ethers, as described inDE-C-4100920, into a state in which it is stable towards digestion byanimal stomachs, in particular the ruminant stomach.

In order to set a desired amino acid concentration in the product, theappropriate amino acid can, depending on the requirement, be addedduring the process in the form of a concentrate or, where appropriate,of a largely pure substance or its salt in liquid or solid form. Thelatter can be added individually, or as mixtures, to the resultingfermentation broth, or to the concentrated fermentation broth, or elsebe added during the drying process or granulation process.

The invention further relates to a process for preparing a solidlysine-containing product (see US 20050220933), which process comprisesprocessing the fermentation broth obtained using the microorganisms ofthe invention in the following steps:

-   a) filtering the fermentation broth, preferably with a membrane    filter so as to obtain a biomass-containing sludge and a filtrate,-   b) concentrating the filtrate, preferably so as to obtain a solids    content of from 48 to 52% by weight,-   c) granulating the concentrate obtained in step b), preferably at a    temperature of from 50° C. to 62° C., and-   d) coating the granules obtained in c) with one or more of the    coating agents).

Preferably, coating agents in step d) are selected from the groupconsisting of

-   -   d1) the biomass obtained in step a),    -   d2) an L-lysine-containing compound, preferably selected from        the group consisting of L-lysine hydrochloride or L-lysine        sulfate,    -   d3) an essentially L-lysine-free substance with an L-lysine        content of <1% by weight, preferably <0.5% by weight, preferably        selected from the group consisting of starch, carrageenan, agar,        silicic acids, silicates, grains, bran and meals, and    -   d4) a water-repellent substance, preferably selected from the        group consisting of oils, polyethylene glycols and liquid        paraffins.

In the case of lysine, the ratio of the ions is preferably adjustedduring the preparation of lysine-containing products such that theequivalent ion ratio in accordance with the following formula2×[SO₄ ²⁻]+[Cl⁻]—[NH₄ ⁺]—[Na⁺]—[K⁺]−2×[Mg²⁺]−2×[Ca³⁺]/[L-Lys]

has a value of from 0.68 to 0.95, preferably of from 0.68 to 0.90, asdescribed by Kushiki et al. in US 20030152633 (The molar concentrationsare to be given in the “[ ]”).

In the case of lysine, the solid fermentation broth-based product whichhas been prepared in this way has a lysine content (as lysine base) offrom 10% by weight to 70% by weight or of from 20% by weight to 70% byweight, preferably of from 30% by weight to 70% by weight and morepreferably of from 40% by weight to 70% by weight, based on the dry massof the product. It is also possible to achieve maximum contents oflysine base of 71% by weight, 72% by weight or 73% by weight.

In the case of an electrically neutral amino acid such as L-tryptophan,the solid fermentation broth-based product which has been prepared inthis way has an amino acid content of at least 5% by weight, 10% byweight, 20% by weight or 30% by weight and maximally 50% by weight, 60%by weight, 70% by weight, 80% by weight, 90% by weight or up to 95% byweight.

The water content of the solid product is up to 5% by weight, preferablyup to 4% by weight, and more preferably less than 3% by weight.

The invention therefore also relates to an L-lysine-containing feedadditive based on a fermentation broth, which has the following features

-   -   a) a lysine content (as base) of at least 10% by weight to no        more than 73% by weight,    -   b) a water content of no more than 5% by weight, and    -   c) a biomass content corresponding to at least 0.1% of the        biomass present in the fermentation broth, wherein the        optionally inactivated biomass is formed by coryneform bacteria        of the invention.

The invention therefore also relates to an L-tryptophan-containing feedadditive based on a fermentation broth, which has the following features

-   -   a) a tryptophan content of at least 5% by weight to no more than        95% by weight,    -   b) a water content of no more than 5% by weight, and    -   c) a biomass content corresponding to at least 0.1% of the        biomass present in the fermentation broth, wherein the        optionally inactivated biomass is formed by coryneform bacteria        of the invention.

The strain MH20-22B was deposited on Oct. 28, 2004 with the DeutscheSammlung für Mikroorganismen und Zellkulturen [German Collection ofMicroorganisms and Cell Cultures] (DSMZ, Brunswick, Germany) as DSM16833.

The Corynebacterium glutamicum mutant DM1915 of the invention, whichcomprises L-leucine in position 38 in the amino acid sequence of the Relpolypeptide, was deposited on May 15, 2006 at the Deutsche Sammlung fürMikroorganismen und Zellkulturen [German Collection of Microorganismsand Cell Cultures] (DSMZ, Brunswick, Germany) as DSM 18257.

The present invention is illustrated in more detail below on the basisof exemplary embodiments.

Example 1

Mutagenesis of the L-Lysine-Producing Strain DM1797

The Corynebacterium glutamicum strain DM1787 was used as parent strainfor mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). StrainDM1797 is an aminoethyl cysteine-resistant mutant of Corynebacteriumglutamicum ATCC13032 and has been deposited under DSM16833 with theDeutschen Sammlung für Mikroorganismen und Zellkulturen (DSMZ,Braunschweig, Germany).

The strain DM1797 was grown in 10 ml of LB broth (Merck, Darmstadt,Germany) in a 100 ml conical flask on a rotary shaker, type CertomatBS-1 (B. Braun Biotech International, Melsungen, Germany), at 33° C. and200 rpm for 24 hours. The culture was then removed by centrifugation,the pellet was resuspended in 10 ml of 0.9% NaCl solution, thesuspension obtained was again removed by centrifugation and the pelletobtained was taken up in 10 ml of 0.9% NaCl solution. 5 ml of this cellsuspension were treated with 400 μg/ml MNNG at 30° C. and 200 rpm onshaker (see above) for 15 minutes. The mutagenesis mixture was thencentrifuged and the pellet was taken up in 10 ml of 2% sodiumthiosulfate in 0.9% NaCl buffer (pH=6.0). The cell suspension was thendiluted with 0.9% NaCl solution in a ratio of 1:1000, 1:10 000 and 1:100000, and aliquots were plated on brain heart agar (Merck, Darmstadt,Germany). Approximately 4500 mutants were isolated in this way.

Example 2

Performance Assay of the DM1797 Strain Mutants

The mutants obtained in example 1 were grown in a nutrient mediumsuitable for producing lysine, and the lysine content in the culturesupernatant was determined.

For this purpose, the clones were firstly propagated on brain heart agarplates (Merck, Darmstadt, Germany) at 33° C. for 24 hours. Starting fromthese agar plate cultures, in each case a preculture was inoculated (10ml of medium in a 100 ml conical flask). The medium used for thepreculture was MM. The preculture was incubated on a shaker at 33° C.and 240 rpm for 24 hours. This preculture was used to inoculate a mainculture, so that the starting OD (660 nm) of the main culture was 0.1.The medium MM was also used for the main culture.

Medium MM CSL 5 g/l MOPS 20 g/l Glucose (autoclaved separately) 50 g/lSalts: (NH₄)₂SO₄) 25 g/l KH₂PO₄ 0.1 g/l MgSO₄ * 7H₂O 1.0 g/l CaCl₂ *2H₂O 10 mg/l FeSO₄ * 7H₂O 10 mg/l MnSO₄ * H₂O 5.0 mg/l Biotin(sterile-filtered) 0.3 mg/l Thiamine * HCl (sterile-filtered) 0.2 mg/lCaCO₃ 25 g/l

CSL (Corn Steep Liquor), MOPS (morpholinopropanesulfonic acid) and thesalt solution were adjusted to pH 7 with aqueous ammonia and autoclaved.The sterile substrate and vitamin solutions and the CaCO₃ autoclaved inthe dry state are added.

Culturing was carried out in volumes of 10 ml each in 100 ml conicalflasks with baffles. The temperature was 33° C., the number ofrevolutions was 250 rpm and the atmospheric humidity was 80%.

After 24 hours, the optical density (OD) was determined at a measurementwavelength of 660 nm with a Biomek 1000 (Beckmann Instruments GmbH,Munich, Germany). The amount of lysine produced was determined using anamino acid analyzer from Eppendorf-BioTronik (Hamburg, Germany) by ionexchange chromatography and post-column derivatization with ninhydrindetection. One mutant distinguished by increased lysine production wasreferred to as DM1915.

TABLE 1 Lysine-HCl Strain OD(660) (g/l) DM1797 9.3 2.2 DM1915 9.2 2.4

Example 3

Sequencing of the Rel Allele of the DM1915 Mutant

Chromosomal DNA was isolated from the DM1915 clone by the method ofEikmanns et al. (Microbiology 140: 1817-1828 (1994)). A DNA sectioncarrying the rel gene or allele was amplified with the aid of thepolymerase chain reaction. Owing to the known sequence of the C.glutamicum rel gene (sequence No. 1824 from EP 1108790), the followingprimer oligonucleotides were selected for the PCR:

rel_XL_A1 (SEQ ID NO: 19): 5′ gcgaattcta tcggatggaa catgaccg 3′rel_XL_A2 (SEQ ID NO: 20): 5′ gcgaattcat gcggatgcca acaagatc 3′

The primers shown were synthesized by MWG Biotech (Ebersberg, Germany)and the PCR reaction was carried out following the standard PCR methodby Innis et al. (PCR Protocols. A Guide to Methods and Applications,1990, Academic Press). The primers enabled an approx. 1.53 kb DNAsection carrying the rel gene or allele to be amplified. Moreover, theprimers comprised the sequence for a restriction site of the EcoRIrestriction endonuclease, which site is underlined in the nucleotidesequence depicted above.

The approx. 1.53 kb amplified DNA fragment carrying the rel allele ofthe DM1915 strain was identified by electrophoresis in a 0.8% strengthagarose gel, isolated from said gel and purified using the usual methods(QIAquick Gel Extraction Kit, Qiagen, Hilden).

The nucleotide sequence of the amplified DNA fragment or PCR product wasdetermined by sequencing by the company Agowa (Berlin, Germany). Thesequence of the PCR product is depicted in SEQ ID NO:15. The sequence ofthe coding region is also depicted in SEQ ID NO:5. The amino acidsequence of the corresponding GTP-pyrophosphate kinase protein, whichwas produced with the aid of the Patentin program is depicted in SEQ IDNO:6.

Position 113 of the nucleotide sequence of the coding region of the relallele of strain DM1915 has the base thymine (SEQ ID NO:5). Thecorresponding position of the wild-type gene has the base cytosine (SEQID NO:1).

Position 38 of the amino acid sequence of the GTP-pyrophosphate kinaseprotein of strain DM1915 has the amino acid leucine (SEQ ID NO:6). Thecorresponding position of the wild-type protein has the amino acidproline (SEQ ID NO:2).

The rel allele which comprises the base thymine in position 113 of thecoding region and correspondingly encodes a GTP-pyrophosphate kinaseprotein whose amino acid sequence comprises the amino acid leucine inposition 38 is referred to below as rel_P38L allele. In the term“rel_P38L”, P is L-proline, L is L-leucine, and 38 indicates theposition of the amino acid substitution (see SEQ ID NO:2 and 6).

The Corynebacterium glutamicum mutant DM1915 which harbors L-leucine inposition 38 of the amino acid sequence of the Rel polypeptide wasdeposited as DSM 18257 with the Deutschen Sammlung für Mikroorganismenand Zellkulturen (DSMZ, Braunschweig, Germany) on 15 May 2006.

Example 4

Substitution of the Rel_P38L Allele for the Rel Wild-Type Gene of StrainDM1797

4.1 Construction of the Replacement Vector pK18mobsacB_rel_P38L

The approx. 1.53 kb DNA fragment described in example 3 and prepared bymeans of PCR, which carries the rel_P38L allele was incorporated intothe chromosome of the C. glutamicum strain DM1797 described in example 1by means of substitution mutagenesis with the aid of the sacB systemdescribed in Schäfer et al. (Gene, 14, 69-73 (1994)). This systemenables allele substitutions occurring by way of homologousrecombination to be produced and selected.

For this purpose, the approx. 1.53 kb rel_P38L fragment was cleaved bythe restriction endonuclease EcoRI, identified by electrophoresis in a0.8% strength agarose gel and subsequently isolated from the gel andpurified using the usual methods (QIAquick Gel Extraction Kit, Qiagen,Hilden).

The mobilizable pK18mobsacB cloning vector was digested with the EcoRIrestriction enzyme and the ends were dephosphorylated with alkalinephosphatase (Alkaline Phosphatase, Boehringer Mannheim, Germany). Thevector prepared in this way was mixed with the approx. 1.53 kb rel_P38Lfragment and the mixture was treated with T4 DNA ligase(Amersham-Pharmacia, Freiburg, Germany).

Subsequently, the E. coli strain S17-1 (Simon et al., Bio/Technology 1:784-791, 1993) was transformed with the ligation mixture (Hanahan, In.DNA Cloning. A Practical Approach. Vol. 1, ILR Press, Cold SpringHarbor, New York, 1989). The plasmid-carrying cells were selected byplating the transformation mixture on LB agar (Sambrook et al.,Molecular Cloning: A laboratory Manual. 2nd Ed., Cold Spring Harbor,N.Y., 1989), supplemented with 25 mg/1 kanamycin.

Plasmid DNA was isolated from a transformant with the aid of the QIAprepSpin Miniprep Kit from Quiagen and examined by restriction cleavage withthe enzyme PstI and subsequent agarose gel electrophoresis. The plasmidis named pK18mobsacB_rel_P38L and is depicted in FIG. 1.

4.2 Allele Substitution

The vector pK18mobsacB_rel_P38L mentioned in example 4.1 was transferredby conjugation into the C. glutamicum strain DM1797 following a protocolby Schäfer et al. (Journal of Microbiology 172: 1663-1666 (1990)). Saidvector cannot self-replicate in DM1797 and is maintained in the cellonly when integrated in the chromosome as a consequence of a recombinantevent. The selection of transconjugants, i.e. of clones containingintegrated pK18mobsacB_rel_Pe8L, was carried out by plating theconjugation mixture on LB agar (Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd Ed. Cold Spring Harbor, N.Y., 1989) supplementedwith 15 mg/l, kanamycin and 50 mg/l nalidixic acid. Kanamycin-resistanttransconjugants were streaked on LB agar plates containing 25 mg/lkanamycin and incubated at 33° C. for 24 hours. Mutants in which theplasmid had been excised as the result of a second recombinant eventwere selected by growing the clones without selection in LB liquidmedium for 30 hours, then streaking them out on LB agar containing 10%sucrose and incubating for 16 hours.

Like the starting plasmid pK18mobsacB, the plasmid pK118mobsacB_rel_P38Lcomprises, in addition to the kanamycin resistance gene, a copy of thesacB gene encoding Bacillus subtilis levan sucrase. Sucrose-inducibleexpression results in the formation of levan sucrase which catalyzes thesynthesis of the product levan which is toxic to C. glutamicum.Therefore only those clones in which the integrated pK18mobsacB_rel_P38Lhas been excised due to a second recombinant event grow on LB agarcontaining sucrose. Depending on the location of the second recombinantevent with respect to the site of mutation, the excision results inallele substitution or incorporation of the mutation or the originalcopy remains in the chromosome of the host.

Approximately 40 to 50 colonies were tested for the phenotype “growth inthe presence of sucrose” and “no growth in the presence of kanamycin”. Aregion of the rel gene, which covers the P38L mutation, was sequenced infour colonies having the “growth in the presence of sucrose” and “nogrowth in the presence of kanamycin” phenotype, starting from thesequencing primer rel-2 (corresponding to the nucleotide sequenceposition 695-714 of the sequence upstream of the CDS of the rel genefrom SEQ ID NO:3), by the company Agowa (Berlin, Germany), in order toverify the presence of the mutation of the rel_P38L allele in thechromosome. For this purpose, the primer used, rel-2, was synthesized byAgowa:

rel-2: 5′ ccg tca ttg tgg tca gag at 3′. (SEQ ID NO: 23)

In this way, a clone was identified which comprises the base thymine inposition 113 of the coding region of the rel gene and therefore has therel_P38L allele. This clone was referred to as DM1797rel_P38L strain.

Example 6

Comparison of the performance of the DM1797rel_P38L strain with that ofthe DM1797 parent strain

The performance assay of the C. glutamicum DM1797rel_P38L strainobtained in example 5 was carried out as described in example 2. Theresult of the experiment is depicted in table 2.

TABLE 2 OD Lysine-HCl Strain (660 nm) g/l DM1797 9.3 2.2 DM1797rel_P38L9.2 2.5

The abbreviations and designations used have the following meaning. Thebase pair numbers given are approximations obtained within thereproducibility of measurements.

Kan: kanamycin resistance gene EcoRI: EcoRI restriction enzyme cleavagesite PstI: PstI restriction enzyme cleavage site rel: rel_P38L allelesacB: sacB gene RP4-mob: mob region with the origin of replication fortransfer (oriT) oriV: origin of replication V

German patent application 102006048882.2, filed Oct. 17, 2006, and U.S.provisional application 60/830,331, filed Jul. 13, 2006, areincorporated herein by reference.

Numerous modification and variations on the present invention arepossible in light of the above teachings. It is therefore to beunderstood that within the scope of the appended claims, the inventionmay be practiced otherwise than as specifically described herein.

1. An isolated polynucleotide encoding an amino acid sequence comprisinga polypeptide having GTP-pyrophosphate kinase enzyme activity, whereinthe polypeptide is as set forth by SEQ ID NO: 2 in which L-proline inposition 38 has been replaced by L-leucine.
 2. The isolatedpolynucleotide as claimed in claim 1, wherein the encoded amino acidsequence is 760 amino acids in length.
 3. The isolated polynucleotide asclaimed in claim 1, which comprises nucleotides 751-3033 of SEQ ID NO:7.
 4. The isolated polynucleotide as claimed in claim 1, whichhybridizes with the nucleotide sequence complementary to the full-lengthof SEQ ID NO: 5 under conditions of hybridization in 5×SSC atapproximately 50° C.-68° C. followed by washing in 0.5×SSC at 66° C.-68°C.
 5. A process for preparing a recombinant microorganism, comprising a)transferring the isolated polynucleotide as claimed in claim 1 to amicroorganism, b) replicating said isolated polynucleotide in saidmicroorganism, and c) propagating the microorganism obtained by a) andb).
 6. A recombinant microorganism comprising the isolatedpolynucleotide as claimed in claim
 1. 7. The recombinant microorganismas claimed in claim 6, which is a coryneform bacterium or a bacterium ofthe genus Escherichia.
 8. The recombinant microorganism as claimed inclaim 7, wherein the coryneform bacterium is the genus Corynebacterium.9. The recombinant microorganism as claimed in claim 8, wherein thebacterium of the genus Corynebacterium is the species Corynebacteriumglutamicum.
 10. A vector comprising the isolated polynucleotide asclaimed in claim
 1. 11. A recombinant microorganism comprising thevector as claimed in claim
 10. 12. The recombinant microorganism asclaimed in claim 11, which is a coryneform bacterium or a bacterium ofthe genus Escherichia.
 13. The recombinant microorganism as claimed inclaim 12, wherein the coryneform bacterium is the genus Corynebacterium.14. The recombinant microorganism as claimed in claim 13, wherein thebacterium of the genus Corynebacterium is the species Corynebacteriumglutamicum.
 15. A process for overexpressing a GTP-pyrophosphate kinasein a microorganism comprising increasing the copy number of thepolynucleotide as claimed in claim 1 in a microorganism by at least one(1) copy.
 16. A process for overexpressing a GTP-pyrophosphate kinase ina microorganism, comprising functionally linking a promotor or anexpression cassette to the polynucleotide of claim 1 and expressing theencoded polypeptide in a microorganism.